Stachybotrys Induced Hemorrhage in the Developing Lung

Date study started January, 1999; Date Study Completed  January, 2004

Recruitment status verified  February, 2001 This study is currently recruiting patients. Sponsored by National Institute of Environmental Health Sciences (NIEHS)

Purpose: The majority of the workscope of this project is focused on an infant animal model of toxigenic mold induced pulmonary hemorrhage. Clinically related studies are directed only to the description of human infants diagnosed with idiopathic pulmonary hemorrhage regarding their clinical parameters, and collecting samples of secretion, blood, and urine for analysis for fungal spores and mycotoxins. These latter analyses are being developed using the infant rat model.

Condition: Idiopathic Pulmonary Hemorrhage

Further Study Details: 

Over the past seven years in the Cleveland, Ohio area there have been 47 cases of unexplained pulmonary hemorrhage in young infants. Sixteen of the infants have died. A CDC case-control study found an association with water-damaged homes and the toxigenic fungus, Stachybotrys chartarum, which requires water soaked cellulose to grow. The spores of this fungus are not infectious in the lung but do contain very potent mycotoxins which appear to be particularly toxic to the rapidly developing lungs of young infants. Secondary stresses, e.g. environmental tobacco smoke, appear to be important triggers of overt hemorrhage. Concern that there may be a larger number of undetected young infants with this disorder led to the examination of all infant coroner cases, which revealed six sudden infant death syndrome cases with major amounts of pulmonary hemosiderin-laden macrophages, indicating extensive hemosiderosis existing prior to death. All of these infants had lived in the eight zip code area where all but fifteen of the patients have lived. This disorder may extend beyond Cleveland since toxigenic fungi are widespread. We are aware of a total of 138 infants with idiopathic pulmonary hemorrhage across the country over the past four years. The purpose of this proposal is to establish an infant animal model for this stachybotryomycotoxicosis which can be used to understand the developmental pathophysiology by which the fungal spores induce hemorrhage and to address practical problems faced in the clinical care of these infants and in public health prevention. The model uses tracheal instillation of toxic Stachybotrys spores in neonatal to weanling rats to initiate the pathological process, followed by stresses to trigger acute hemorrhage. Capillary fragility to transmural pressures will be assessed by morphometric analysis of electron micrographs. Markers of Stachybotrys exposure which can be applied to clinical cases are being developed and tested in the rats.

Dorr G Dearborn, MD,PhD, dxd9@po.cwru.edu

The Immunopathology of Hypersensitivity Reactions

Vincent A. Marinkovich, M.D. Clinical Associate Professor, Stanford Medical School, Redwood City, CA

Abstract

Overactivity of the immune system, either allergy (IgE) or hypersensitivity (non-IgE) is responsible for more illness than is generally appreciated, even by the medical profession. The least understood are the non-IgE mechanisms which involve either immune complex formation (type III of Gell and Combs) or direct killer T-cell involvement (type IV). Type III reactions may be localized with a large deposition of antigen at a focal point where immune complexes are formed and tissue damage ensues including necrosis. This is termed the Arthus reaction. A systemic dissemination of antigens will provoke a systemic inflammatory reaction which is most closely modeled by the well studied acute and chronic serum sickness reaction. Serum sickness was identified as the constellation of symptoms which followed the administration of antitoxins (antiserum given for infectious disease before the advent of antibiotics) which were derived from non-human sources, most often horses. Chronic serum sickness was observed when otherwise healthy subjects were given repeat doses of antiserum experimentally over relatively short periods of time. The symptoms observed in spontaneous and experimental serum sickness included fatigue, rash, cognitive changes, myositis, arthritis, headache, weight-loss, cardiovascular symptoms etc., which are often seen during heavy chronic exposure to fungal spores. The dynamic nature of circulating immune complexes, their complexity, their rapidly changing equilibrium patterns and their pathogenicity must be appreciated before the clinician can properly interpret the patterns of illness his patients describe. The best simple test identifying and thereby allowing the avoidance of serum sickness is a specific IgE test to a panel of high exposure antigens including fungi, food and occupational antigens.

Hypersensitivity Pneumonitis from Toxic Mold Exposure

Fungal Spores: Hazardous to Health? W.G. Sorenson

National Institute for Occupational Safety and Health, Morgantown, West Virginia USA

Fungi have long been known to affect human well being in various ways, including disease of essential crop plants, decay of stored foods with possible concomitant production of mycotoxins, superficial and systemic infection of human tissues, and disease associated with immune stimulation such as hypersensitivity pneumonitis and toxic pneumonitis. The spores of a large number of important fungi are less than 5 µm aerodynamic diameter, and therefore are able to enter the lungs. They also may contain significant amounts of mycotoxins. Diseases associated with inhalation of fungal spores include toxic pneumonitis, hypersensitivity pneumonitis, tremors, chronic fatigue syndrome, kidney failure, and cancer. Key words: mold, fungi, mycotoxin, lung disease, toxic pneumonitis. -- Environ Health

Cognitive Impairment Associated with Exposure to Toxigenic Fungi

Wayne A. Gordon, Ph.D., Eckardt Johanning, M.D.,M.Sc. Lisa Haddad, B.A. Professor, Department of Rehabilitation Medicine, Mount Sinai School of Medicine; Eckardt Johanning, M.D., Adjunct Instructor, Department of Community Medicine, Mount Sinai School of Medicine; Lisa Haddad, B.A., Research Assistant, Department of Rehabilitation Medicine, Mount Sinai School of Medicine; Research performed at Mount Sinai School of Medicine, Correspondence: Wayne A Gordon Ph.D., Department of Rehabilitation Medicine, Mount Sinai School of Medicine

The objective of the study was to examine whether cognitive impairment was associated with exposure to Stachybotrys atra. 20 individuals were examined using a standard neuropsychological battery and a symptom checklist. Results indicate that all individuals seen meet at least 1 of the criteria for a cognitive impairment, with 13 of the 20 (65%) meeting at least 3 of the criteria for cognitive impairment. Preliminary findings suggest that exposure to toxigenic molds are associated with cognitive impairment, specifically deficits in verbal memory, verbal learning, attention/concentration and set shifting. In addition to these cognitive deficits, the sample reported a high number of cognitive, physical and behavioral symptoms. Validation on a larger sample of individuals is indicated.

Prevention and Treatment of Skin Lesion of T-2 Toxin

Author: Dr. A. Khaboshany, Dr. A. Omidi, Dr. S.M. Morsali

Institution: Medical Science University, Mashhad

Trichothesin mycotoxins are a group of fungal metabolites which are produced by the same species of fusarium. These compounds have a common structure and depending on various substitutions on the ring, different toxins with special function are produced. These toxins cause many cellular damages in contaminated men and animals. These damages will appear in animal organs which have grown faster. For example, most damages will cause to bone marrow, skin and digestive tract cells. These toxins react with - SH group of enzyme. For example, peptidy transpheras, which have a role important in the synthesis of cellular proteins and inhibited action of the enzymes. In this investigation, we chose compounds for prevention and treatment which react with toxins and compete in their reaction with - SH group of enzymes. Thiol compounds were the best and from these four Thiol compounds, N- Acetylcysteine, sodium hyposulfatl, cysteine hydrochloride and menthionine were chosen and their effects in prevention and treatment. Application of NAC pomad to the contaminated position after 30 min., decreased 50-70 percent of lesions. Also, application of pomad after 60 min., decreased 40-60 percent of lesions.

Building-Related Illness in Occupants of Mold-Contaminated Houses

Toxic Mold Exposure- Case Study Investigation

James Craner, M.D., M.P.H. Consultant in Occupational & Environmental Medicine, Verdi, Nevada, Assistant Clinical Professor, Department of Medicine, Division of Occupational Medicine, University of California, San Francisco School of Medicine, Address for correspondence: P.O. Box 1161, Verdi, NV 89439

The clinical presentation and course of individuals and families who developed illnesses related to non-infectious fungal exposures inside their homes is described. Occupants developed their illnesses shortly after their homes had been water damaged. A few occupants had a specific building-related illness, such as hypersensitivity pneumonitis or asthma exacerbation, but most had a "sick building syndrome" symptom complex involving irritation/inflammation of the mucous membranes, respiratory tract, and skin; fatigue; and/or neurocognitive dysfunction. All cases required months or years to correctly diagnose. Air, surface, and/or bulk microbiological sampling in most of the homes yielded high concentrations of toxigenic fungi, including Stachybotrys chartarum and Penicillium and Aspergillus species, emanating from water-damaged building materials. Most of the ill individuals had complete clinical improvement shortly after their removal from the contaminated indoor environment, but a few individuals continued to experience symptoms in response to a variety of environmental irritants. The author proposes a new clinical syndrome entity to describe the non-infectious, mold-related, building-related illness.

Diagnosing the Cause of a "Sick Building:"

Case Study of an Epidemiological and Microbiological Investigation

James Craner, M.D., M.P.H., Linda d. Stetzenbach, Ph.D. Consultant in Occupational & Environmental Medicine, Verdi, Nevada Director, Department of Microbiology, Harry Reid Center for Environmental Studies, University of Nevada-Las Vegas, Las Vegas, Nevada

This report describes the methods and outcome of a physician-led investigation of occupants' prolonged, unexplained illnesses associated with working inside a large, modern office building. Occupants (cases) complained of building-related symptoms including eye, nose, and throat mucous membrane irritation; rashes; respiratory symptoms; profound, unexplained fatigue; and neurocognitive symptoms, including difficulty concentrating and short-term memory impairment. No functional ventilation problems or chemical contamination were detected in a walk-through evaluation and basic air quality testing. An epidemiological survey of the building's 700 occupants was then conducted. With 86% of the occupants responding to the survey, there was an average case prevalence of health-related complaints of nearly 25%, evenly distributed among floors. Cases were geographically distributed in a pattern which coincided with the location of the ceiling-mounted variable air volume (VAV) boxes which distributed ventilated air to the occupied spaces. Re-inspection revealed previously undetected, focal water-staining of 40% of ceiling tiles located underneath the VAV boxes throughout the building. Active growth of Stachybotrys chartarum (atra) and other fungi was detected on many of the damaged tiles. All water-damaged tiles were replaced and VAV hot water valves were tightened. Occupants reported significant improvement of symptoms within weeks after these changes

Effects of Mycotoxins on Human Immune Functions in Vitro

Berek L, Petri IB, Mesterhazy A A, Teren J, Molnar J.

Immunosuppressive and carcinogenic Fusarium mycotoxins may appear in domestic food products. Therefore, the immunological effects of Fusarium mycotoxins were tested on human peripheral blood mononuclear cells from different blood donors. In the present study we investigated deoxynivalenol (DON), 3-acetyldeoxynivalenol, fusarenon-X, T-2 toxin, zearalenone, alpha-zearalenol, beta-zearalenol and nivalenol for their effects on T and B cells in a proliferation assay, antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell activity on human peripheral blood mononuclear cells. The concentrations applied in our experiments were similar to those which can be found in normal human peripheral blood system (0.2-1800 ng/ml). Among the eight mycotoxins tested, T-2 toxin, fusarenon X, nivalenol and deoxynivalenol exerted the highest immunosuppressing effect on human peripheral blood mononuclear cells in vitro. Mycotoxin-induced immunosupression was manifested as depressed T or B lymphocyte activity. Furthermore, by virtue of inhibition of NK cell activity, the protection against tumor development may also be attenuated.

Agonistic and Antagonistic effects of Zearalenone, an Etrogenic Mycotoxin, Human Cancer Cell lines

Withanage GS, Murata H, Koyama T, Ishiwata I.

Zearalenone (ZEA) is a nonsteroidal estrogenic compound mainly produced by the molds Fusarium graminearium and Fusarium culmorum found in a variety of host plants and soil debris around the world. ZEA is usualy non-lethal to animals but is important to livestock producers because its hyperestrogenic effects adversely influence the reproductive performance of animals. There have been suggestions of possible involvement of ZEA in the progression of breast malignancies and tumors of the female reproductive tract in humans. The toxic or stimulatory effects of ZEA and its metabolites alpha-zearalenol and 17-beta-estradiol on SKN, HHUAand HepG2 cells were studied using rapid colorimetric MTT assay. In general, both concentrations of 17-beta-estradiol (100M and 10 nM) were toxic to SKN and HHUA cell cultures. Both ZEA and alpha-zearalenol stimulated the proliferation of SKN and HHUA cells. On HepG2 cells, lower concentrations (10 nM) of 17-beta-estradiol and higher concentrations (100 microM) of ZEA exhibited toxic effects, whereas treatment with higher concentrations of 17-beta-estradiol and lower concentration of ZEA did not show toxic effects. A dose dependent antagonistic effect was observed when the cell cultures were pre-incubated with ICI 182,780, a synthetic estrogen receptor blocker, before estradiol or mycotoxin treatments.

Trichothecenes

This Page is part of the CU Toxic Plant Pages and was constructed by Yuan-Kuo Chen.

Trichothecenes are produced by several common molds including species in the genera Acremonium (Cephalosporium), Cylindrocarpon, Dendrodochium, Myrothecium, Trichoderma, Trichothecium, and most numerously in Fusarium. Trichothecenes are composed of a tetracyclic sesquiterpene skeleton containing a six-membered oxane ring, a stable epoxide group in positions 12 and 13 and a 9,10 olefinic bond. They have been classified into four groups. Fusarium spp. contain several well known trichothecenes including two highly toxic members of group A, diacetoxyscirpenol (DAS) and T-2 toxin, and toxins in group B including deoxynivalenol (DON) and nivalenol. DON is the most common but least toxic of these. Trichothecenes are strong inhibitors of protein synthesis in mammalian cells. However, DOM received its common name, vomitoxin, from the vomiting that generally accompanies trichothecene poisoning. (D'Mello, et.al., 1991)

DON resulted in feed refusal in swine. In lambs, consumption of a wheat diet containing DON at 15.6 mg/kg of BW for 28 d did not alter feed consumption, weight gain, or feed efficiency. Oral administration of DON showed that it was rapidly passed essentially unchanged (95%) and excreted primarily in urine. Incubation of DON with ruminal microorganisms in vitro for 48 h resulted in partial conversion to deepoxy DON. These results indicate that the impact of DON on ruminants is lower than initially suspected. DON caused no organ damages to animals. Extremely low amounts of DON(<4ng/ml) were transmitted to milk after a single oral dose of 920 mg to a dairy cow. (Diekman and Green, 1992)

The FDA issued an "advisory" to federal and state officials recommending a level of concern for DON of 2 micrograms of DON/ gm for wheat entering the milling process, 1 microgram/ gm in finished wheat products for human consumption, and 4 microgram/ gm for wheat and wheat milling by-products used in animal feed. (Wood, 1992)

Toxigenic Fungi: Which are Important?

Pitt JI.

Growth of commonly occurring filamentous fungi in foods may result in production of mycotoxins, which can cause a variety of ill effects in humans, from allergic responses to immunosuppression and cancer. According to experts, five kinds of mycotoxins are important in human health around the world: aflatoxins, ochratoxin A, fumonisins, certain trichothecenes, and zearalenone. These toxins are produced by only a few species of fungi, in a limited range of commodities. Aflatoxins are potent carcinogens, produced by Aspergillus flavus and A. parasiticus in peanuts, maize and some other nuts and oilseeds. Ochratoxin A is a kidney toxin and probable carcinogen. It is produced by Penicillium verrucosum in cereal grains in cold climates, by A. carbonarius in grapes, wines and vine fruits, and by A. ochraceus sometimes in coffee beans. Fumonisins, which may cause oesophageal cancer, are formed by Fusarium moniliforme and F. proliferatum, but only in maize. Trichothecenes are highly immunosuppressive and zearalenone causes oestrogenic effects; both are produced by F. graminearum and related species. Current reporting probably underestimates the effect of mycotoxins as a cause of human mortality.

Toxigenic Fungi and Mycotoxins

Pitt JI.

Growth of commonly occurring filamentous fungi in foods may result in production of toxins known as mycotoxins, which can cause a variety of ill effects in humans, from allergic responses to immunosuppression and cancer. The most important mycotoxins are aflatoxins, ochratoxin A, fumonisins, trichothecenes and zearalenone. Aflatoxins are potent carcinogens and, in association with hepatitis B virus, are responsible for many thousands of human deaths per annum, mostly in non-industrialised tropical countries. Ochratoxin A is a probable carcinogen, and may cause urinary tract cancer and kidney damage in people from northern and eastern Europe. Fumonisins appear to be the cause of oesophageal cancer in southern Africa, parts of China and elsewhere. Trichothecenes are highly immunosuppressive and zearalenone causes oestrogenic effects in animals and man. Currently available records and statistics do not reflect the major role played by mycotoxins in mortality attributable to food-borne micro-organisms.

Exposure Biomarkers in Chemoprevention Studies of Liver Cancer

Wild CP, Turner PC.

Molecular Epidemiology Unit, Epidemiology and Health Services Research, School of Medicine, University of Leeds, UK.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer, the major risk factors being hepatitis B and C viruses and aflatoxins; other factors such as alcohol are also of importance in some populations. Aflatoxin exposure biomarkers include urinary aflatoxin metabolites and aflatoxin-albumin adducts in peripheral blood. These biomarkers are well validated and have been applied in studies of many populations worldwide. They are proving to be valuable end-points in intervention studies, including chemoprevention studies. The biomarkers permit assessment of primary prevention measures to reduce aflatoxin intake. In addition, the determination of individual urinary aflatoxin metabolite profiles means that the effectiveness of chemopreventive agents designed to modulate aflatoxin metabolism can also be evaluated. Both aflatoxin-albumin adducts and urinary aflatoxin metabolites have been associated with increased HCC risk in prospective studies, indicating the predictive value of these biomarkers at the group level. However, given the multifactorial and multistep nature of HCC, it is unlikely that these exposure biomarkers will be predictive at the individual level or be of value as surrogate end-points in longer-term intervention trials aimed at reducing disease incidence. Aflatoxin-related mutations at codon 249 of the p53 gene in plasma may be more relevant in this regard but their application requires further understanding of the temporal appearance of this biomarker in relation to the natural history of the disease.

Mechanisms of Aflatoxin B1 Lung Tumorigenesis

Massey TE, Smith GB, Tam AS.

Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario, Canada. masseyt@post.queensu.ca

Although aflatoxin B1 (AFB1) is best known as a hepatocarcinogen, the respiratory system can also be a target of this mycotoxin. In isolated lung cells from rabbits and mice, AFB1 is bioactivated by cytochromes P450, primarily in nonciliated bronchiolar epithelial (Clara) cells. However, mutagenesis experiments suggest that the DNA-binding AFB1 epoxide metabolite can leave the cells of origin, and potentially interact with other cell types. Consistent with DNA adduct studies, AFB1-induced AC3F1 mouse lung tumors contain point mutations at guanine residues in K-ras, with the anticipated bias for the A/J allele. Furthermore, following AFB1 treatment but prior to tumor development, K-ras mutations occur preferentially in mouse Clara cells. However, in contrast to findings with other carcinogens, AFB1-induced mouse lung tumors demonstrate frequent, but heterogeneously distributed, overexpression of p53 protein as well as p53 point mutations, suggesting a carcinogen-specific response. Unlike lung tissue from mice and rabbits, human peripheral lung bioactivates AFB1 primarily by prostaglandin H synthase--and/or lipoxygenase-catalyzed cooxidation, with activity concentrated in macrophages. In addition, although glutathione S-transferase M1-1 has high specific activity for AFB1 epoxide conjugation, lung tissues from GSTM1-null individuals do not demonstrate diminished rates of conjugation, compared to tissues from GSTM1-positive individuals. In summary, AFB1 tumorigenesis in mice demonstrates unique properties, and processes of bioactivation show significant species differences.

Relationship Between Lung Cancer and Aflatoxin B1

Georggiett OC, Muino JC, Montrull H, Brizuela N, Avalos S, Gomez RM.

Catedra de Cirugia II-UA Cirugia H. Misericordia Fac. Cs. Medicas U.N.C.

The relationship between aflatoxins and liver cancer is well established. In addition the inhalation exposure to carcinogen aflatoxin B1 (AFB 1) is considerable. Genotoxic chemical is known to react with DNA either directly or after metabolic activation to form adducts, a step thought to be relevant with respect to chemical carcinogenesis. The presence and the amount of specific DNA adducts provide a good indication of chemical exposure and genetic damage resulting the exposure to carcinogens and account for same of factors affecting individual susceptibility to cancer. Analysis of DNA adducts requires that the sensitivity of the methods to be sufficient high to allow detection of about 1 adduct/109 normal nucleotides. Most suitable method is based in physiochemical technique such as HPLC. Because circumstantial epidemiological evidence suggests that AFB1 inhalation may cause primary lung cancer. We investigate AFB1 by HPLC in three different tobacco sources, and in 39 patients with compatible lung cancer or chronic bronchitis. The patients were divided by clinical manifestations in lung cancer (n: 25) and chronic bronchitis (n: 14). Twenty-three of 25 patients presented epidermoid lung cancer within smoking habit, and 2 of 25 presented adenocarcinoma without smoking habit. In chronic bronchitis group 12 of 14 cases presented smoking habit. The control PBS liquid was negative to AFB1; the different tobacco sources, a) Virginia of Jujuy, b) Brasilero and c) black of Salta presented AFB1 positive determinations respectively. The bronchial tissues obtained by lung biopsies presented positive AFB1 in lung epidermoid cancer at 0.68 +/- 0.82 mg/L. The adenocarcinoma presented AFB1 negative determinations. In chronic bronchitis patients with smoking habit (n: 12) presented AFB1 positive with a level less than the epidermoid lung cancer group, 0.21 +/- 0.109 mg/L, p < .025.

Neuronal Effects of Microbial Toxins

Project leader Kai Savolainen, Department of Industrial Hygiene and Toxicology

Topeliuksenkatu 41 aA, FIN-00250 Helsinki, Finland Researchers H. Stockmann

Completed by May 2002

Collaborating parties Finnish Research Programme on Environmental Health, Kuopio University

Objectives: Fungal growth in moisture problem houses often leads to exposure to microbes which produce microbial toxins. Besides the typical respiratory tract disorders, recent findings suggest that microbial toxins may cause serious effects in the central nervous system of animals and man. The objective of the study is to try to explore the mechanisms whereby the toxins affect neuronal cells.

Subjects: Neuroblastoma and glioblastoma cells.

Methods: Studying the effects of microbial toxins on oxidarive stress, transcription factors and cell viability (necrosis and apoptosis). Fluorescent probes and different gel separation methods.

Effect of Individual Metabolic Characteristics on Genotoxicity

Project leader Hannu Norppa, Department of Industrial Hygiene and Toxicology Helsinki, Finland. Researchers S. Bernardini, G. Falck, A. Hirvonen, H. Järventaus, J. Tuimala

Completed by Dec. 2005

Objectives: The goal of the project is to clarify the influence of metabolic polymorphisms on the genotoxicity of chemicals in humans.

Materials: The importance of genetic polymorphisms is studied among smokers, foundry workers, bus drivers exposed to vehicle exhaust and nuclear power plant employees, and also among control persons. In addition, the genotoxic metabolites of some industrial chemicals are examined in lymphocyte cultures of donors representing different genotypes.

Methods: The genotoxicological methods applied include cytogenetic analyses from peripheral lymphocytes. Genotype determinations are carried out by means of techniques based on polymerase chain reaction.

Health Effects, Pathology, Epidemiology

Effects after Mold Exposure – Which are the Causative Agents?

Ragnar Rylander, M.D. e-mail: ragnar.rylander@envmed.gu.se

This presentation reviews the most well known specific agents in molds and their relation to the different effects caused by exposure to molds. It is emphasized that field studies in principle only describe relationships and that such relationships not always support conclusions regarding causality for individual agents. At present, the general strategy to prevent the mold-related diseases airways inflammation, increased risk for infections, hypersensitivity pneumonitis and neurological disorders must therefore be to remove the mold contamination, viable as well as dead organisms.

Ecology, Detection and Identification Problems of Moulds in Indoor Environments

Robert A. SAMSON Ph.D.

Centraalbureau voor Schimmelcultures, The Netherlands, e-mail: samson@cbs.knaw.nl

For many people working in the building industry or those who are consulting for indoor problems, identification of moulds does not seem a problem. In their opinion mould applying disinfectants or antifungal paints can easily solve problems. Furthermore, it is often recommended to modify the construction and adding ventilation with the result that the conditions for fungal growth can be reduced. However, if we like to know the cause and the degree of biodeterioration, in particular when health implications are involved, it is important to know which organisms are causing the problem. This is particularly true for species of Stachybotrys, Penicillium, Aspergillus and Fusarium.

Identification of mould isolates or specimens is problematic for the inexperienced researcher. This is partly due to the fact that the methods for identification are still based on the phenotypical characters of the moulds, which requires many skills in microscopical observations. In addition the mould flora of indoor environments consists of some problematic genera such as Penicillium, Aspergillus and Fusarium of which there is no general taxonomic consensus. Even experienced mycologists might reach different conclusions hence different names for an isolate.

Several surveys of moulds in indoor environments have revealed that we are dealing with a known and relatively low number of species. To detect these relevant species it is, however, essential to apply the appropriate methods. This paper is focused on the ecology and taxonomic problems of indoor moulds also related to methods of detection.

Occupational Exposure to Molds, Diseases and Diagnosis

Kari Reijula, M.D., Ph.D.

Director, Indoor Air & Environment Program; Finnish Institute of Occupational Health; Finnish institute of Occupational Health, email: krei@occuphealth.fi

Exposure to molds has become a significant health risk to an increasing number of workers in various occupations throughout the nations. Fungal antigens are able to cause occupational asthma, rhinoconjunctivitis, hypersensitivity pneumonitis and organic dust toxic syndrome (ODTS) for instance among farmers, construction workers, housekeeping personnel as well as factory and sawmill workers who have to handle moldy and contaminated materials. In recent years, an increasing incidence of mold-induced diseases has been encountered in moldy contaminated water-damaged buildings. This has occured both in homes and workplaces. Symptomatic persons occupying moisture problem buildings may develop asthma, rhinitis, ODTS and HP. However, the majority of the exposed individuals present with conjunctival, respiratory or systemic reactions the immune mechanisms of which remain unclear. In the present summary some aspects will be presented on health effects which appear among workers exposed especially to fungal antigens.

Indoor Moulds: a Public Health Problem in Belgium

Nicole Nolard, Ph. D.

Introduction

This paper is a summary of highlights recorded from 15 years’ experience of surveys in home environments of patients with respiratory disorders linked with allergy, mainly asthma. Actually, after the 1st oil crisis, in the 1970’s, people began to renovate their house with the aim of best insulating every part from cellar to attic and we registered a continuing increase in complaints correlated with the presence of fungi, not only in damp houses but also in renovated and even new houses. Each survey responded to a specific case with different situation standards (house/flat; city / country, underprivileged / luxurious; pets / no pets) Moreover, taking into account the evolution of the sampling methods, a standard methodology was rather difficult to elaborate. Under these circumstances, in 1982, a scheme was set up in our laboratory at the Institute of Public Health, which belongs to the Belgian Ministry of Health, for environmental control in homes. It includes:

a visit to the home

a standardized home environment form,

the sampling of air, surfaces, furniture, wall paper, mattress and carpet dust for fungal moulds

the isolation, purification and placing in our collection of fungal strains for immunological testing (more than 2000 strains are stored either freeze dried or under liquid nitrogen in the IHEM collection),

the creation of a serum bank containing not only the serum of the allergic patient but also the sera of people living in the same surroundings,

the standardisation of a mini-method for preparing fungal extracts from selected strain,

finally, the immunological analysis from allergic patients, from subjects exposed to the same environment but not symptomatic and from a pool of test sera. Prick tests and antibody research (IgE and IgG) are carried out, or are in the process of being carried out, with our own extracts and have confirmed the role, in greater and greater numbers, of moulds as pneumoallergens in the home. Our objective is to present in this article a synthesis of our work at the Institute and to inform about the interest of environmental surveys.

Pilot Analysis of the Immune Response to Fungal Antigens in Subjects Working in Humidity Damaged Houses

Professor of Clinical Immunology and Microbiology e-mail: Anja.Tiilikainen@oulu.fi

Abstract

Although the proportion of anti-mold IgG antibody producers was largely the same among different groups of subjects tested, the highest OD-readings in EIA technique were seldom seen in symptomless controls, but generally in association with any allergy of the "mold exposed" subjects or in patients referred to specialist analysis at the Oulu University Hospital outpatient clinic for oto-rhino-laryngology. Some subjects in each group were distinctly multi-immune ("responders" = R) and some just occasionally reactive ("low responders" = LR) against a panel of 12-16 mold antigens; of the 34 allergic subjects 6 were R and 3 LR. In vitro cultures of patient lymphocytes usually responded with a lower cytokine production to Aspergillus fumigatus antigen than to other mold antigens, in contrast to responses of lymphocytes from healthy controls. More often than not, R lymphocytes showed higher cytokine production than did LR lymphocytes; more frequently in healthy controls than in patients.

Can Microbial Volatile Metabolites Cause Irritation at Indoor Air Concentrations?

University of Kuopio, Department of Environmental Sciences, Kuopio, Finland; Corresponding author: Prof. Anna-Liisa Pasanen; University of Kuopio, Department of Environmental Sciences, email: annal.pasanen@uku.fi

Sensory irritation potency of selected volatile organic compounds assumed to be of microbial origin (MVOCs) was estimated theoretically by using the data of the recent chamber experiments and the results of volatile organic compounds (VOCs) measurements in water-damaged buildings. According to the estimations, a possible risk for complaints of irritation caused by exposure to the mixtures of those MVOCs in moisture problem buildings was evaluated. Exposure to mixtures of the selected non-reactive VOCs at the theoretical airborne concentrations calculated from the data of the chamber experiments would not result in sensory irritation in humans, and, thus, microbial growth in constructions should not increase the probability of irritating symptoms considerably. The data on MVOC concentrations measured in some problem buildings also supported this hyphothesis. Irritation would be expected when the airborne concentrations of single assumed MVOC approach a level of hundreds of m g/m3 or mg/m3.

Growth Conditions of Streptomyces Anulatus Regulate Induced Inflammatory Responses and Cyto Toxicity in Macrophages.

Division of Environmental Health, National Public Health Institute, Division of Environmental Health Maija-Riitta Hirvonen, Ph.D, National Public Health Institute, email Maija-Riitta.Hirvonen@ktl.fi

Abstract

RAW264.7 macrophages were exposed for 24 hrs to the spores of Streptomyces anulatus (doses of 105 106, 107 spores/million cells), isolated from a moldy building and grown on 23 different kind of media. Production of nitric oxide (NO) in macrophages was induced dose dependently by spores of S. anulatus reaching the level from 4.2 mM to 39.2 mM depending on the composition of the medium. The expression of inducible NO synthase (iNOS) was detected in the macrophages after the exposure to the spores collected from all tested growth media. The production of reactive oxygen species (ROS) was significantly increased when compared to controls by the highest dose of the spores from two media. The growth media affected the production of cytokines as well; the highest dose dependent levels of IL-6 varied from 400 to 7500 pg/ml and the levels of TNF a from 450 to 3100 pg/ml. The amount of death macrophages after the exposure varied from 11 % to 98% depending on the growth media of the microbe. Altogether, our results suggest that the growth conditions of S. anulatus, have a fundamental role in the ability of spores to induce inflammatory responses and cytotoxicity in RAW264.7 cells.

Clinical Findings Related to Indoor Fungal Exposure - Review of Clinic Data of a Specialty Clinic

Eckardt Johanning M.D., M.Sc., Paul Landsbergis Ph.D. johanni2@crisny.org

Objective: Review of symptoms, clinical abnormalities and laboratory findings of building occupants with abnormal indoor fungal exposure, primarily allergenic and toxigenic fungi (Stachybotrys atra, Penicillium, Aspergillus).

Design: Descriptive review of 151 cases evaluated at a specialty clinic. Analyses of a standardized health symptom questionnaire and immunological laboratory data.

Subjects: Pediatric and adult specialty clinic population with verified abnormal indoor fungal exposure.

Main outcome measures: Self-reported symptom-complexes, Immunoglobulin E and G, T- lymphocyte enumeration and function.

Results: Widespread contamination of water-damaged building materials primarily with toxin producing fungi were identified in the patients exposure history, who are reporting from a variety of nonspecific"sick building"-like complaints to a history of more serious morbidity of the respiratory system, skin, eye, chronic fatigue-like symptoms (CFIDS) and central nervous system dysfunctions (vertigo, memory, irritability, concentration, verbal dysfunctions). In some cases abnormalities of enumeration and functional laboratory tests (flow-cytology), mainly of the white blood cell system were identified. IgE or IgG antibodies used as exposure markers, were positive in about less than 25% of all cases. Removal from exposure typically resulted in improvement in the majority of cases.

Conclusion: Indoor air exposure to mycotoxin and allergen producing fungi results in a high frequency of health complaints, variant multi-organ and laboratory abnormalities requiring a detailed exposure assessment and clinical evaluation. Removal from fungal exposure and symptomatic treatment generally results in noticeable improvement of most patients. I propose to name these presentations and clinical findings-if certain criteria are met: "fungal syndrome".

Pulmonary Hemorrhage Among Infants with Exposure to Toxigenic Molds

Ruth A. Etzel1, M.D., Ph.D., Dorr G. Dearborn2, Ph.D., M.D.

Case Western Reserve University, Cleveland, Ohio 44106

In 1994, we investigated a geographic cluster of 10 cases of acute idiopathic pulmonary hemorrhage among infants in Cleveland, Ohio. Our matched case-control study demonstrated that infants with this condition were more likely than control infants to live in homes with toxigenic Stachybotrys chartarum and other fungi in the indoor air. The risk appeared to increase when both S. chartarum and environmental tobacco smoke were present in the home. Since the completion of the 1994 investigation, 27 additional infants in the Cleveland area have been diagnosed with acute idiopathic pulmonary hemorrhage. Of the 37 infants, 30 were African-American infants, all of whom lived in a limited geographic area of eastern metropolitan Cleveland, an area of older housing stock. Twelve deaths with extensive idiopathic pulmonary hemosiderosis have been identified, including seven originally thought to be due to Sudden Infant Death Syndrome (SIDS).

To determine how common this problem might be, we actively solicited reports of idiopathic pulmonary hemorrhage in infants from pediatricians throughout the United States. We have received physician reports of an additional 101 cases of acute idiopathic pulmonary hemorrhage among infants under 1 year of age. This paper reviews the risk factors for this condition and discribes the ongoing surveillance efforts.

IAQ and Human Toxicosis: Empirical Evidence and Theory

Harriet M. Ammann, Ph.D., D.A.B.T.

Washington State Department of Health, e-mail: HMA0303@doh.wa.gov

Studies of injury, illness and death occurring in mold-exposed animals and people in the field, observe that the illness called mycotoxicosis results from more complex exposures than can be observed in laboratory experiments with pure mold toxins. Response in field exposures occurs at lower exposure concentrations than those from controlled experiments. Occurrence of signs and symptoms at low exposure levels may result from a number of factors, including a greater spectrum of susceptibility in heterogeneous populations, additive or synergistic actions of multiple toxins, actions of other toxic microbial compounds, allergic and irritant response, as well as concomitant exposure to other contaminants. Changes in the immune system, often reflected as increased susceptibility to infectious illness, are a common finding of low level exposure to toxigenic molds that inhibit protein synthesis. Changes in the immune system are extremely complex, but changes in the endocrine and nervous system accompany them, and may reflect changes in the central neuroendocrine-immune control system. Evaluation of illness of individuals exposed to a combination of irritant, allergenic and toxigenic biological contaminants in wet buildings requires more information about basic effects of individual agents as well as interactive effects of the complex mixtures. In the meantime, case definition for such illness needs to include exposure parameters that actually reflect the nature of the agents capable of effecting illness, rather than some isolated component such as a single mycotoxin. A definition of mycotoxicosis borrowed from agricultural animal and worker exposures, "illness resulting from exposure to one or more toxic molds and their products," could serve. A case definition should also include location of individuals relative to the location of suspect contaminants, time of contact with or exposure to molds and their products, and a list of signs and symptoms gleaned from investigations of known animal or human toxic exposures. Commonality of signs and symptoms in those who show similar time and place exposure parameters could be used to further focus definition of a case. Prudent public health practice recognizes the potency of the toxic agents produced by toxigenic molds, and seeks to protect occupants of buildings once moisture incursion with resultant microbial growth has been discovered.

Cognitive Impairment Associated with Exposure to Toxigenic Fungi

Wayne A. Gordon, Ph.D., Eckardt Johanning, M.D.,M.Sc. Lisa Haddad, B.A.

The objective of the study was to examine whether cognitive impairment was associated with exposure to Stachybotrys atra. 20 individuals were examined using a standard neuropsychological battery and a symptom checklist. Results indicate that all individuals seen meet at least 1 of the criteria for a cognitive impairment, with 13 of the 20 (65%) meeting at least 3 of the criteria for cognitive impairment. Preliminary findings suggest that exposure to toxigenic molds are associated with cognitive impairment, specifically deficits in verbal memory, verbal learning, attention/concentration and set shifting. In addition to these cognitive deficits, the sample reported a high number of cognitive, physical and behavioral symptoms. Validation on a larger sample of individuals is indicated.

Symptoms Associated to Work in a Water Damaged School Building

T.Sigsgaard MD.,PhD., H.L.C.Jensen B.Sc., E.Nichum B.Sc, S.Gravesen M.Sc., L.Larsen M.Sc., M..Hansen B.Sc. Copenhagen, Environmental E-mail: TS@mil.au.dk

This paper describes a cross sectional study of employees from a school, where an annex had a long history of water damage. The annex had infestations by a range of moulds with Penicillium, Aspergillus, Cladosporium most often encountered. The employees filled in a questionnaire with questions on sick building syndrome extended with symptoms of toxic alveolitis. After allocating the employees into three groups according to the weekly hours spent in the annex of 0-7 h/week, 8-15h/week and more than 15 hours/week. We found a positive trend for headache, tiredness, nausea and sleeping difficulties with increasing time spent in the annex. This was also true for episodes of fever, shivering or a flue-like feeling. This study emphasises the need for a standardised way to perform investigations of water damaged buildings and indicates a dose response relationship between exposure to water damaged buildings and symptoms of inflammation and CNS-irritation even in low exposure situations.

Sensory Irritation of Microbially Produced Volatile Organic Compounds in Mice During Repeated Exposures

Anne Korpi, PH. LIC., Jukka-Pekka Kasanen, M.Sc., Anna-Liisa Pasanen, Ph.D. Department of Environmental Sciences, Kuopio, Finland; email: anne.korpi@uku.fi

Microbially produced volatile organic compounds (MVOCs) are suspected to cause eye, nose and throat irritation in occupants of moldy buildings. The effect of repeated exposures (30 minutes per day during 4 consecutive days) of mice to 3-octanone (3531 mg/m3), 1-octen-3-ol (36 mg/m3), or to a mixture of five MVOCs (58 mg/m3) via inhalation was studied with a standardized method (ASTM E 981-84). With single MVOCs, no changes in the responses between repetitions of exposure were seen, and only a very slight adaptation in the respiratory response was noted along with the repetition of exposure to a mixture of MVOCs. Thus, during a short-term experiment, repeated exposure to MVOCs did not provoke changes in the sensation of irritation nor cause permanent effects on upper respiratory tract.

Immunological Biomonitoring in the Assessment of Exposure to Airborne Fungi from Waste Handling

J. Bünger1, M.D., M. Müller, Ph.D., K. Stalder, M.D., Prof., E. Hallier, M.D., Prof.

Center of Environmental and Occupational Medicine, Department of Occupational and Social Medicine, Georg-August-University, Göttingen, Germany e-mail: ehallie@gwdg.de

The immunologic reaction of workers in different waste treatment facilities to inhalation of mold antigens was studied by determination of specific IgG antibody levels and compared to exposures. Antigens were prepared from cultures of fungi which were dominant in dust samples at these workplaces. In 520 waste workers, 32 patients with symptoms of hypersensitivity pneumonitis (HP), and 98 control subjects, serum concentrations of specific IgG antibodies to antigens of molds (Aspergillus and Penicillium species) were determined by an indirect immunofluorescence test (IIFT). The highest antibody levels were found in workers in compost plants, followed by employees of garbage sorting facilities. Lower levels were determined in workers at landfills and lowest in collectors of household biowaste. This order reflects the ranking according to the exposure measurements. About 5% of workers had elevated antibody levels, but none complained about typical symptoms of HP. The IIFT proved to be a very sensitive method of antibody detection since even IgG levels of persons subjected to environmental exposure alone (control) could be measured. High exposure to organic dust at workplaces with waste handling is correlated with elevated mold-specific IgG antibody levels.

Chronic Toxic Encephalopathies Apparently Related to Exposure to Toxigenic Fungi

Pierre L. Auger M.D., Pierrot Pépin H.I., J. David Miller Ph.D., Manfred Gareis D.V.M., Ph.D., Julien Doyon Ph.D., Rémi Bouchard M.D., Marie-France Pinard Ph.D., Claude Mainville Ing.

This report concerns four cases of neurotoxic encephalopathies possibly caused by a prolonged exposure to toxin producing filamentous fungi (molds) in the workplace. We will also demonstrate that complete fungal evaluation with species identification and complementary cytotoxicity testing can be useful to identify possible causes of health effects of mold exposures.

Building-Related Illness in Occupants of Mold-Contaminated Houses

James Craner, M.D., M.P.H.

Consultant in Occupational & Environmental Medicine, Verdi, Nevada,

The clinical presentation and course of individuals and families who developed illnesses related to non-infectious fungal exposures inside their homes is described. Occupants developed their illnesses shortly after their homes had been water damaged. A few occupants had a specific building-related illness, such as hypersensitivity pneumonitis or asthma exacerbation, but most had a "sick building syndrome" symptom complex involving irritation/inflammation of the mucous membranes, respiratory tract, and skin; fatigue; and/or neurocognitive dysfunction. All cases required months or years to correctly diagnose. Air, surface, and/or bulk microbiological sampling in most of the homes yielded high concentrations of toxigenic fungi, including Stachybotrys chartarum and Penicillium and Aspergillus species, emanating from water-damaged building materials. Most of the ill individuals had complete clinical improvement shortly after their removal from the contaminated indoor environment, but a few individuals continued to experience symptoms in response to a variety of environmental irritants. The author proposes a new clinical syndrome entity to describe the non-infectious, mold-related, building-related illness.

Diagnosing the Cause of a "Sick Building:" a Case Study of an Epidemiological and Microbiological Investigation

James Craner, M.D., M.P.H., Linda d. Stetzenbach, Ph.D.

Consultant in Occupational & Environmental Medicine, Verdi, Nevada

Director, Department of Microbiology, Harry Reid Center for Environmental Studies, University of Nevada-Las Vegas, Las Vegas, Nevada

This report describes the methods and outcome of a physician-led investigation of occupants’ prolonged, unexplained illnesses associated with working inside a large, modern office building. Occupants (cases) complained of building-related symptoms including eye, nose, and throat mucous membrane irritation; rashes; respiratory symptoms; profound, unexplained fatigue; and neurocognitive symptoms, including difficulty concentrating and short-term memory impairment. No functional ventilation problems or chemical contamination were detected in a walk-through evaluation and basic air quality testing. An epidemiological survey of the building’s 700 occupants was then conducted. With 86% of the occupants responding to the survey, there was an average case prevalence of health-related complaints of nearly 25%, evenly distributed among floors. Cases were geographically distributed in a pattern which coincided with the location of the ceiling-mounted variable air volume (VAV) boxes which distributed ventilated air to the occupied spaces. Re-inspection revealed previously undetected, focal water-staining of 40% of ceiling tiles located underneath the VAV boxes throughout the building. Active growth of Stachybotrys chartarum (atra) and other fungi was detected on many of the damaged tiles. All water-damaged tiles were replaced and VAV hot water valves were tightened. Occupants reported significant improvement of symptoms within weeks after these changes.

Fungal Exposure and IGg-levels of Occupants in Houses with and without Mold Problems

Hyvärinen A., M.Sc., Reiman M., Ph.D., Meklin1 T, M.Sc., Husman T., M.D, Vahteristo M, M.Sc., MD., Nevalainen A, Ph.D.

National Public Health Institute, Email: Anne.Hyvarinen@ktl.fi

The aim of the study was to evaluate the relevance of serum antifungal-IgG antibodies to reflect the individual´s exposure to building related fungi. The concentrations of viable fungi were higher and composition of fungal flora was different in the index houses with moisture problem compared to those in the reference houses. Positive IgG-findings were common in both groups, although higher antibody levels against most of the fungi were found in the study group than in the control group. However, compatibility between fungal flora found in each house and elevated IgG-levels of its occupants were seldom found in either of the groups. This study suggests that microbial sampling gives information of the building at the time of the measurements, whereas IgG-antibodies reflect long-term integrated exposure of an individual from total environment including the work place.

The Immunopathology of Hypersensitivity Reactions

Vincent A. Marinkovich, M.D.

Clinical Associate Professor, Stanford Medical School,

Overactivity of the immune system, either allergy (IgE) or hypersensitivity (non-IgE) is responsible for more illness than is generally appreciated, even by the medical profession. The least understood are the non-IgE mechanisms which involve either immune complex formation (type III of Gell and Combs) or direct killer T-cell involvement (type IV). Type III reactions may be localized with a large deposition of antigen at a focal point where immune complexes are formed and tissue damage ensues including necrosis. This is termed the Arthus reaction. A systemic dissamination of antigens will provoke a systemic inflammentory reaction which is most closely modeled by the well studied acute and chronic serum sickness reaction. Serum sickness was identified as the constellation of symptoms which followed the aadministration of antitoxins (antisera given for infectious disease before the advent of antibiotics) which were derived from non-human sources, most often horses. Chronic serum sickness was observed when otherwise heaalthy subjects were given repeat doses of antisera experimentally over relatively short periods of time. The symptoms observed in spontaneous and experimental serum sickness included fatigue, rash, cognitive changes, myositis, arthritis, headache, weight-loss, cardiovascular symptoms etc., which are often seen during heavy chronic exposure to fungal spores. The dynamic nature of circulating immune complexes, their complexity, their rapidly changing exquilibrum patterns aaand their pathogenicity must be appreciated before the clinican can properly interpret the patterns of illnes his patients’ describe. The best simple test identifying and thereby allowing the avoidance of serum sickness is a specific IgE test to a panel of high exposure antigens including fungi, food and occupational antigens.

Exposure to Stachybotrys Chartarum Induces Immunoglobulin a Antibody Response in Man

Päivi Raunio M.Sc, Anna Pasanen Prof., Tuula Husman M.D., Tuomas Virtanen M.D. University of Kuopio, e-mail: Paivi.Raunio@uku.fi

The levels of serum immunoglobulin (Ig) E, G and A antibodies against Stachybotrys chartarum were measured in patients with asthmatic or mycotoxicosis symptoms (n=6), their family members (n=10) and control subjects (n=18) with indirect enzyme-linked immunosorbent assays. The Stachybotrys-specific IgG and IgA levels correlated with each other significantly (r=0.71, p<0.01) in the group of exposed subjects (patients and family members), but not in the control group. The IgA levels were significantly higher (p<0.01) and the IgG levels slightly higher (p<0.05) in the patient group than in the control group. IgE levels did not differ between the subject groups. The results of this study suggest that the exposure to Stachybotrys does not cause IgE-mediated allergy in humans. It is possible that the IgA response reflects better exposure to the fungus than the IgG response does.

Sensitization to Molds and Respiratory Symptoms in School Children

Taskinen1 T., M.D., Hyvärinen A., M.Sc., Meklin T., M.Sc., Husman T., M.D., Nevalainen A., Ph.D., Korppi1 M. M.D.

Kuopio University Hospital, E-mail: Taina.Taskinen@ktl.fi, taina.taskinen@kiuruvesi.fi

We performed a questionnaire study in 622 school children; asthma or asthmatic symptoms were present in 208 (33%) children. Moisture and mold problems weredocumented in the index school (N=414; 168 (41%) were symptomatic); the control school (N=208; 40 (19%) were symptomatic, p<0.001) had no such problems. Skin prick tests to 13 molds (9 occurring in environment and 4 indicating moisture problems in buildings) were performed in the 208 symptomatic children. A positive reaction (>3mm) was observed in only 5 (2%) children. Children who were exposed to molds in the school had more often emergency visits (OR=2.0, p<0.01) and also they had used more antibiotic courses (OR=2.1, p<0.01) than nonexposed pupils, but only during spring. Our results show that skin test positivity to molds is rare in school children. This is true in pupils with asthmatic symptoms, and also in the pupils from the school with moisture or mold problems. In addition, respiratory infections seem to be associated with mold exposure.

Mycotoxin Cytotoxicity Screening of Field Samples

Manfred Gareis, D.V.M., Ph.D., Prof., Eckardt Johanning, MD., M.Sc. Ritchie Dietrich, Ph.D. Institute for Microbiology and Toxicology, e-mail: gareis.baff@t-online.de

Several hydrophil fungi produce very potent chemical metabolites (mycotoxins) that may pose a particular health risk in indoor settings. However, these properties have not been routinely assessed in similar field investigations, due to a lack of readily available screening test methods. A total of 166 moulded samples of building materials (gypsum boards, wallpaper, ceiling tiles, fiberglass and other insulating materials, carpets, dust and air filters) from 44 case investigations conducted during the time period from 1993 to 1998 in various areas of the United States were tested with the MTT-cytotoxicity screening assey for the presence of cytotoxic mycotoxins and compared with controls. A subset of samples was analysed by an enzyme-immune assay (EIA) for occurrence of macrocyclic trichothecenes produced by strains of Stachybotrys chartarum. In addition, presence of fungi were examined by culture methods and scanning electron microscopy. Different levels of cytotoxicity were detected in particular of field samples of gypsum board or wall paper visibly contaminated with fungal growth. Samples with highly cytotoxic effects reacted positive in the MTT-bioassay at concentrations of less than 5 mg of sample aliquot per ml of cell culture medium and indicated the presence of cytotoxic contaminants. Twentythree field samples (52%) of all case investigations were positive for cytotoxicity compared with controls. Most frequently detected fungi in toxic samples were Stachybotrys chartarum (85%), Trichoderma sp. (31%) and Chaetomium sp. (38%) compared to non-cytotoxic samples with a proportion of 38%, 10% and 5%, respectively. These results indicated the presence of non-toxigenic strains or alternatively the non-production of cytotoxic mycotoxins by toxigenic strains on these particular samples. The level of toxicity obtained by the MTT-bioassay highly correlated with the amount of macrocyclic trichothecenes detected by use of the EIA in the samples contaminated with or without Stachybotrys chartarum. Toxicity detected in samples not contaminated with Stachybotrys chartarum indicates the presence of cytotoxic mycotoxins produced by other fungi. In conclusion, the results support the usefulness of the MTT cytotoxicity assay as an important diagnostic tool for the assessment of potential mycotoxin exposure inside buildings with fungal contamination.

The Effect-Inhaled Spores of Mycotoxin Producin Fungi on Animals

Eeva-Liisa Hintikka, D.V.M., Ph.D.

Head of the Department of Bacteriology, National Veterinary and Food Research Institute, Helsinki, FINLAND

Animals in contact with a mouldy environment are exposed to respiratory fungal infection, lung mycosis. Toxic fungal spores in the air create a risk of respiratory effect. Animal experiments have shown that pure mycotoxins administered to the respiratory organs have only a transient or no effect in the lungs. Toxin-containing spores of Stachybotrys atra administered to the respiratory organs of mice cause severe haemorrhages and lung inflammation.

Trichothecenes as a Potent Inducer of Apoptosis

Hiroki Okumura, Naoto Yoshino, Yoshitsugu Sugiura, Masao Sugamata, Eeva-liisa Hintikka, Bruce Jarvis, Yoshio Ueno

Department of Toxicology and Microbial Chemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Ichigaya, E-mail youeno@kagu.sut.ac.jp

Induction of apoptosis by the trichothecenes was examined in promyelotic leukemia cell line HL-60 and mice. An early elevation of intracellular Ca++ ion followed by disruption of observed mitochondrial transmembrane potential, induction of caspase-3, and DNA fragmentaion was oberved in T-2 toxin-treated cells. An induction of apoptotic cell death in the tissues of mice given T-2 toxin was confirmed by electron microscopic analysis. The induction of apoptosis was proposed as a novel cell death mechanism of T-2 toxin as well as macrocyclic trichothecenes.

Analysis for Stachybotrys Toxins

Bruce B. Jarvis1, Ph.D., Prof., Simon F. Hinkley

Department of Chemistry and Biochemistry, and Joint Institute for Food Safety and Nutrition, University of Maryland, College Park, MD e-mail address: bj6@umail.umd.edu

Analytical conditions have been developed for the detection and quantitation of several classes of mycotoxins produced by the toxigenic mold, Stachybotrys atra (S. chartarum). Fungal cultures or environmental samples are extracted with methanol-chloroform and the crude extracts passed through a PEI silica cleanup column. The fractions from this cleanup column are analyzed by reversed phase chromatography (C-18, acetonitrile-water-formic acid) coupled to a diode array detector. Three major classes of mycotoxins are produced by S. atra: trichothecenes, phenylspirodrimanes, and, a new class of diterpenoids, the atranones. Thirty-eight isolates of S. atra were assayed for their mycotoxin production, and the S. atra isolates fell into two distinct classes: those that produce trichothecenes (12/38) and those that produce atranones (22/38).

Assessing Bioaerosols in Elementary School Classrooms

K.H. Bartlett, Ph.D., S.M. Kennedy, Ph.D., M. Brauer, Sc.D., B. Dill, Ph.D., C. VanNetten, Ph.D. K.H. Bartlett, Ph.D kbartlet@interchange.ubc.ca

A two year study of elementary school classrooms was begun in 1996. Data collected included: number of occupants and patterns of occupancy, CO2 levels, temperature and percent relative humidity (%RH), total particulate, and air exchange rates using tracer gas (SF6). Indoor and outdoor bioaerosols were collected using an Andersen N-6 impactor.

The effect of measured indoor air quality parameters and building characteristics on the airborne concentrations of indoor bacteria and mesophilic fungi were evaluated using multiple linear regression. The variable with the highest predictive value for bacterial concentrations was indoor CO2 concentration, with the final equation having a R2 of 0.59. In contrast, outdoor temperature had the highest predictive value for indoor mesophilic fungi with the final equation having a R2 of 0.61.

Variables commonly measured during an indoor air quality investigation can be used to understand bioaerosol concentrations. High commensal bacterial counts are clearly related to inadequate ventilation of the occupied space. In a similar manner, the use of mechanical or natural ventilation in buildings and the concentration of fungi in the outdoor environment is related to indoor fungal counts. These data would suggest that indoor air quality investigations benefit from an integrated investigative approach.

Anatomy of a Fungal Problem

Neil Carlson1, M.S., C.I.H., Arif Quraishi2, B.S.M.E., M.B.A.

University of Minnesota, 410 Church Street S.E., Minneapolis

Sampling was performed in a school with extensive visible fungal growth to evaluate exposure potential to building occupants. Musty odors and allergy-type symptoms were reported by building occupants. Initial testing using fungal sampling was not successful in identifying risks to building occupants. Additional sampling was conducted using total spore trap sampling, tape sampling and microbial volatile organic compound (MVOC) sampling. Analysis of the samples using total spore trap and tape sampling methods identified exposure potential to Acremonium spp., Alternaria spp., Paecilomyces spp., Aspergillus niger and others. Measurement of MVOCs showed that the indoor MVOCs were almost double the outside level. While sampling performed by traditional viable methods suggested no exposure potential, tape, spore trap and MVOC sampling data revealed greater exposure potential. Based on the results of this study, viable fungal sampling should not be the only method for conducting risk assessments relating to exposure to fungal spores and their bioeffluents.

Prevalence of Fungi in Carpet Dust Samples

Mark Hodgson LRSC, Richard Scott AIA

Senior Consultant, Occupational Health and Safety; Clayton Environmental Consultants, mail: Mhodgson@claytongrp.com or Rscott@Claytongrp.com

Samples of dust collected from carpeting can provide an indication of the microbial condition of a building with the carpet material having potential to be both an amplifier (growth site) and reservoir for fungi. Interpretation of the data obtained from analysis of the dust is helpful when making a determination as to the overall condition of both the building and the carpet material. The authors have collated data from 243 samples collected in both problem and control buildings across the USA. A comparison is made between the results obtained from both problem and control buildings, with particular emphasis on the biodiversity found and the regional differences observed. The study concludes that despite the ubiquitous nature of fungi there are a number of key indicators which can be used to determine if the dust from a carpet suggests a contaminated building.

Detection and Decontamination of a Facility Contaminated with Fungi Including Stachybotrys chartarum

Ling-Ling Hung, Ph.D. US Public Health Service, Division of Federal Occupational Health, Philadelphia, PA Email: lhung@foh.dhhs.gov

A facility indoor environmental quality investigation was initiated in 1996 due to onset of employees' health symptoms after fifteen minutes exposure in the facility. The investigation was to determine the extent of contamination, identify the causative agents, design decontamination specifications, and develop and implement a monitoring strategy for clearance sampling.

Massive fungal growth was observed on various surfaces. Many types of microbiological samples were collected throughout this two-story facility. Very high fungal levels were detected on bulk and wipe samples (107 CFU/g and 106 CFU/in2). Three rounds of air samplings were conducted. Outdoor fungal levels were at 102 CFU/m3 with Cladosporium as the predominant fungi. However, indoor fungal levels were consistently at least 104 CFU/m3. Indoor fungal flora was dominated by Aspergillus, Penicillium, and Stachybotrys chartarum. Analytical results showed that basement, ground floor, and ductwork were contaminated.

Specifications were developed to decontaminate the facility and the ductwork. Ten percent (10%) bleach solution and Oxine® brand of chlorine dioxide (500 ppm) was used as the disinfectant for the facility and the ductwork, respectively. Final clearance samples were taken to evaluate the effectiveness of the cleaning. Air sampling results showed that airborne fungal levels in the facility were compatible to those of outdoors (230 and 224 CFU/m3, respectively), with a similar fungal flora. Results from final clearance surface samples, taken after completion of second ductwork cleaning, showed that surface fungal burden of this facility was not different from that of a reference building.

Airborne Concentrations of Trichoderma and Stachybotrys linked to Mycotoxicosis

Larry D. Robertson, M.S., B.S. Mycotech Biological, Inc., Jewett, TX

The affected individual developed symptoms approximately 55 days after exposure to a working environment containing significant fungal contamination. Initial symptoms included bronchitis, swelling, spastic colon, severe headaches, and fatigue. Later, abdominal pain, nausea, diarrhea, and loose teeth were reported. Three physicians having specialties in internal medicine, toxicology, and neurology independently diagnosed exposure to an "unknown" environmental toxin; however, the physicians were not provided with environmental results from the work place. The work environment indicated airborne concentrations of Trichoderma viride and Stachybotrys chartarum at 494 CFU/m3 and 212 CFU/m3, respectively. Active Trichoderma viride and Stachybotrys chartarum growth sites were documented at levels of 3.3 x 104 CFU/g and 2.0 x 107 CFU/g; respectively. Although these contaminants were exclusive to the individuals work area, the CIH/CSP represented the data as "typical" for indoor environments. As a result, no exposure data was provided to the attending physicians. After 5 months of exposure the individual became too ill to return to work. Within 1 month of removal the symptoms begin to subside. Complete symptom cessation resulted after 12 months. The absence of specific human dose-exposure data relative to the various mycotoxins produced by Trichoderma viride and Stachybotrys chartarum does not support a valid medical claim relative to an aerosol-induced mycotoxicosis. However, anecdotal, circumstantial, and environmental information strongly supports the potential for this event to have occurred. This study demonstrates the current void that exists relative to both the knowledge and availability of mycotoxin diagnostic methods in the medical field and suggests the immediate need for education, training, and research relative to aerosol generated mycotoxicoses.

Exposure Measures for Studies of Mold and Dampness and Respiratory Health

J. David Miller M.Sc., Ph.D., Robert Dales M.D., M.Sc. Jim White, P. Eng. Department of Chemistry, Carleton University, Ottawa, Ontario.

This paper reviews some basic principles underlying the measurement of fungi in air samples and dust to describe their quantitative and qualitative value. We compare the results of such measurements with our experience in the Wallaceburg study wherein ca. 400 homes in southern Ontario were studied for various exposure and health outcomes. Until more powerful methods to determine quantitative and qualitative exposures to fungi are developed, the attributable risk of fungal contamination in residential housing for public health cannot be estimated with accuracy. The most reliable information on mold exposures in residential housing can be obtained by measuring moisture source strength, detailed study of areas of visible mold plus some mycological testing of building materials to determine where the growth is occurring beyond that visible to the naked eye and qualitative and quantitative analysis of molds in settled dust.

Fungal Growth in Buildings: The Aerobiological Perspective

Harriet A. Burge Ph.D. Harvard School of Public Health, Boston,

In the search for inexpensive shelter, we have developed indoor environments that are conducive to fungal contamination. While active fungal growth indoors is usually inappropriate and should be controlled, assessing specific health risks associated with such growth remains a challenge. Epidemiological tools are often used to determine relative risks associated with occupancy by groups of people in environments with or without certain factors, including fungal growth, but do not always make clear the role of the growth in the disease process in indiviudals. Aerobiologists assess relationships along a pathway that includes sources, dispersion and decay of aerosols, exposuree to individuals, doses of agents, and responses. Both approaches yield valuable information, but require the development of testable hypotheses.

As a model, we can apply the epidemiological and aerobiological processes to the Cleveland hemosiderosis outbreak, and consider the following hypotheses:

The Cleveland babies that develop hemosiderosis are more likely to live in moldy homes than those that did not develop the diseease, all other things being equal.

The Cleveland babies that developed hemosiderosis are more likely to live in homes with Stachybotrys than those that did not develop the disease, all other things being equal.

Aerobiology Babies that developed disease were likely to have received a dose of Stachybotrys chartarum toxins sufficient to cause the reported symptoms.

Some evidence exists to support Hypothesis 1.

Hypothesis 2 is suppored by very little evidence

Hypothesis 3 has not been tested.

Until Hypotheses 2 and 3 areadequately tested and verified, assuming a cause/effect relationshipfor Stachybotrys toxins in these cases is premature. Premature establishement of cause/effect relationships may lead to unneccessaary conceern, and prevent discovery of actual caauses of disease. This hypothesis development and testing process is essential if we are to accurately determine the role of indoor fungi in human disease.

Why are there Still Problems with Fungal Allergen Extracts? 

W. Elliott Horner, Samuel B. Lehrer Air Quality Sciences, Inc., Atlanta, GA, and Tulane University Medical Center, New Orleans, LA email aqs@mindspring.com

It has long been axiomatic that reliable allergen extracts of fungi are more difficult to produce and work with than extracts of pollen, dander, or other allergen sources (Salvaggio, 1981; Burge, 1985; D’Amato, Spieksma, 1995; Feinberg, 1946). Features of fungi that contribute to this include their number and variety, the identification is sometimes difficult, their phenotypic plasticity, and the presence of endogenous proteases that can degrade allergens in crude extracts. All of these problems can be addressed to an extent, albeit some with difficulty. Indeed, with the few fungi with which molecular techniques are being used, recombinant allergens are readily being produced. Unfortunately, adequate resources and resolve are lacking for a concerted effort at producing well-characterized allergen extracts. Further, the extracts that are available correspond poorly with the fungi that are often a problem indoors.

Comparative studies of fungal media for the recovery of Stachybotrys Chartarum from Environmental Samples

Stella M. Tsai1, M.Sc., Chin S. Yang, Ph.D., Patricia Heinsohn, Ph.D., CIH P&K Microbiology Services, Inc., Cherry Hill, NJ

Five commonly used fungal media were used to determine the relative recovery efficiencies of Stachybotrys chartarum (SC) by culturing sixty-five unknown environmental samples from building materials. These fungal media were cornmeal agar (CMA), Czapek cellulose agar (CCA), 2% malt extract agar (2% MEA), 1% malt extract agar (1% MEA) and rose bengal agar (RBA). The samples were first examined microscopically for the presence of SC. It was found that all five fungal media were all suitable for the recovery of SC from the environmental samples. The recovery frequency of SC from bulk samples ranged from 87.7% on 2% MEA to 95.4% on CMA. Qualitative differences of colony growth and interaction between S. chartarum and other fungi were observed on the media. CMA yielded the best sporulation and highest recovery rate of SC from the environmental samples. The other four media also supported the isolation and recovery of SC but at reduced rates.

Heteroduplex DNA Fingerprinting of Penicillium Brevicompactum from House Dust

James A. Scott, Neil A. Straus1, Bess Wong Department of Botany, University of Toronto E-mail: jscott@sporometrics.com

It is widely believed that many osmotolerant microfungi, including toxigenic species of Aspergillus and Penicillium proliferate on indoor substrata such as dust and broadloom even in the absence of appreciable moisture. This hypothesis has been offered as an explanation for the disproportionate abundance of propagules of these species in indoor environments, relative to their representation in outdoor reservoirs such as air and soil.

We obtained several thousand isolates of Penicillium from 367 homes in southwestern Ontario, Canada. From these, 75 isolates of P. brevicompactum representing 54 houses were selected based on micromorphological and physiological uniformity. Sequences of PCR-amplification products of polymorphic genetic loci were compared between isolates using heteroduplex mobility assay (HMA), demonstrating two primary, genetically divergent groups which appear to be stable, clonally-reproduced lineages within the dust mycoflora. These clones are distributed throughout the sample population and co-exist at several sites, suggesting that in absence of objective moisture problems, the principal amplifiers of P. brevicompactum lie outside the building environment. It is possible that mechanical / filtration effects (e.g. differential removal of propagules by vacuum cleaning and elutriation in air conveyance systems), and the relatively long spore viabilities of trichocomaceous anamorphs play a significant role in concentrating propagules of these microfungi in homes.

The Trichodiene Synthase Gene from Stachybotrys Chartarum : A Potential Diagnostic Indicator of Indoor Contamination

Neil A. Straus PhD, James Scott, Bess Wong MSc Department of Botany, University of Toronto, Canada Email: straus@botany.utoronto.ca

The hyphomycete Stachybotrys chartarum readily grows on damp cellulosic areas in buildings producing spores that may contain highly toxic trichothecenes. Here we report the cloning, sequencing of the gene for trichodiene synthase which is the first enzyme of the pathway uniquely dedicated to the synthesis of trichothecenes. The predicted amino acid sequence shows regions of high conservation. Nucleotide sequence divergence permits the selection of PCR primers that can detect the trichodiene synthase gene of S. chartarum in DNA diagnostic strategies.

Microscopic Fungi and Metabolites in Dwellings-a Bioassay Study

Ing. Elena Piecková, M.P.H., Ph.D., MUDr. Zdenka Jesenská, Dr., Sc., Ken Wilikins, Ph.D. Institute of Preventive and Clinical Medicine, Bratislava, Slovakia

The ciliostatic effect of chloroform-extractable endo- and exocellular metabolites of the most frequently isolated fungal strains from growth in dwellings: Alternaria sp., Aspergillus glaucus group, A. versicolor, Cladosporium sphaerospermum, Penicillium sp., P. chrysogenum, Stachybotrys chartarum, Trichoderma viride and Ulocladium sp. was studied on tracheal cilia from day old chicks in vitro. Biomass extracts from Alternaria sp. and A. versicolor as well as exocellular extracts from P. chrysogenum, S. chartarum I, T. viride and Ulocladium sp. stopped cilia movement during the first 24 hours. Biomass extracts of P. chrysogenum, S. chartarum I and T. viride as well as media extracts from Alternaria sp., C. sphaerospermum and Penicillium sp. stopped cilia movement between 24 and 48 hours. Between 48 and 72 hours the biomass extract of an A. glaucus gr. isolate and media extracts of A. versicolor and S. chartarum II showed activity while the other extracts (cellular from C. sphaerospermum, S. chartarum II and Ulocladium sp. and media one from A. glaucus gr.) showed no activity. The results are discussed in relationship with health status of people living/working in moldy buildings.

Moisture, Mold and Health in Apartment Homes

A. Nevalainen, Ph.D., M. Vahteristo, M.Sc., J. Koivisto, Civ.Eng., T. Meklin, M.Sc., A. Hyvärinen, M.Sc., J. Keski-Karhu, M.Sc., T. Husman, M.D. National Public Health Institute, Division of Environmental Health e-mail aino.nevalainen@ktl.fi

The prevalence of observations of moisture or mold, and the respiratory health status of the occupants, was surveyed in a random sample of 120 apartment buildings. The buildings and two apartments from each were given a walk-through inspection and all the signs of moisture and mold recorded using questionnaires and checking lists by civil engineers. A health questionnaire was sent to the occupants. In 60% of the apartments, signs of moisture damage could be observed, and 42% of the apartments were assessed to be in need of repair because of the moisture observations. Respiratory symptoms, such as cough, nocturnal cough and dyspnea, sore throat, hoarseness, rhinitis, nasal bleeding and impaired sense of smell were significantly associated with the observations of moisture, as well as sinusitis (OR=2.58). When the exposure was defined as mold present the symptom findings were almost similar. The results show the health-based importance of good maintenance.

Toxigenic Microbes in Indoor Environment: Identification, Structure and Biological Effects of the Aerosolizing Toxins

Salkinoja-Salonen M.S., Ph.D., Andersson M.A., M.Sc, Mikkola R., M.Sc, Paananen A.,B.Sc, Peltola J., M. Agr. & For., Mussalo-Rauhamaa M.D.,Ph.D., Saris N Ph.D., Grigorjev Ph.D, Helin Ph.D., Koljalg, Ph.D, Timonen M.D.,Email: mirja.salkinoja-salonen@helsinki.fi

Almost 500 pure cultures, bacteria and fungi, were isolated and identified to genus or species level from indoor environments where the occupants were suffering health problems. Over 80 different taxa were identified. Seven bacterial species classified to Hazard Group 2 were found. Extracts prepared from the building materials and from the pure cultures, were tested for their effects on cellular energy production and conservation using boar spermatozoon and human NK cells as test cells. Strains giving toxic responses were found among strains from 11 taxa. Cell free extracts prepared from cultures of Bacillus cereus, Bacillus licheniformis, Streptomyces griseus, a new species of Nocardiopsis, Stachybotrys chartarum and Trichoderma sp were shown to contain toxins that depleted the spermatozoon of ATP and destroyed plasma membrane integrity. Strains of Streptomyces griseus and Bacillus cereus were shown to produce toxins that caused mitochondrial swelling and programmed NK-cells towards apoptosis. These toxins were dodecadepsipeptides and operated as K+ ionophores across the mitochondrial membrane. Strains of Bacillus licheniformis and Trichoderma produced toxins that depleted the spermatozoon of ATP. The toxins were nonenzymatic, highly hydrophobic, robust molecules of small size (<104 g mol-1), effective at low concentration (ppb). We propose that the building related health symptoms of the occupants exposed to one or several of the taxa indicated above, may be caused by microbially emitted toxins. We discuss possible mechanisms of human toxicity.

Evaluation of Exposure to Environmental Bacteria

Laitinen Sirpa, Ph.D., Kangas Juhani, Ph.D. Kuopio Regional Institute of Occupational Health, Finland

The present study provides information about the methods used to determine airborne bacteria in occupational environments. The best means proved to be the analysis of filter samples with endotoxin and peptidoglycan assays. The results of the assay, which measures the biological activity of cell wall components, mainly endotoxins, correlated well with the measured concentrations of viable airborne bacteria, especially with the levels of gram-negative bacteria. The endotoxin analyzed by chemical markers did not correlate well with the results of the assay nor with the viable bacteria levels, despite the similarity between the bacterial species identified from the culture media and the corresponding 3-OH fatty acids analyzed by the GC-MS assay. Indicative information on total bacteria levels could be obtained from peptidoglycans, which correlated well with total viable bacteria.

Cellular and Humoral Responses in an Animal Model Inhaling Penicillium Chrysogenum Spores

J. Danny Cooley, Ph.d., Wing C. Wong, M.S., Cynthia A Jumper, M.D., David C. Straus, Ph.D. Departments of Microbiology and Immunology1 and Medicine at Texas Tech University Health Sciences Center, Lubbock, Texas

Penicillium chrysogenum (Pc) is a potential causative agent of the complaints and symptoms of occupants in buildings experiencing "sick building syndrome". Viable Pc spores were recovered from the lungs of mice 15 minutes and 3 hours through 36 hours after intranasal (IN) inoculation of 1x106 spores, of which 25% were viable. Eighteen percent of the viable spores were deposited in the lungs, however, by 12 h, only 1x104 viable spores were recovered. This suggests that the mucociliary tract had cleared the majority of spores deposited, but four percent (1x104) of the viable spores were retained in the airways and were probably deposited in the alveolar spaces and remained viable for up to 36 h post-inoculation. Similar acute doses of viable spores induced significant (P<0.001) increases in tumor necrosis factor a (TNF-a), while non-viable (NV) Pc spores did not. Repeated doses (3 weeks) of 1x104 viable spores induced significant (P<0.05) increases in total serum IgE and bronchioalveolar lavage (BAL) interleukin-4 (IL-4), whereas 1x104 NV spores did not. This suggests that viable Pc spores are capable of inducing allergic responses.

Sporulation of the Hyphomycete Stachybotrys chartarum Under Three Light Conditions

Patricia Heinsohn1, Ph.D., C.I.H., Sharon Harney, Ph.D., K. Alexandros Exuzides, Ph.D. Menlo Park, CA

Stachybotrys chartarum is a widespread hyphomycete commonly isolated from a variety of substrates including soil and wood. It can be found growing in building materials, which have become wet before or after construction. The inhalation of S. chartarum conidia can cause pneumomycotoxicoses, and a recent study linked S. chartarum to the deaths of infants diagnosed with pulmonary hemosiderosis in Cleveland. While S. chartarum is frequently isolated, the conditions under which it sporulates are unknown and are important issues in assessing indoor air quality. This study reports on the sporulation of S. chartarum under three different artificial light conditions. Two isolates of S. chartarum were inoculated onto two different media, MEA and CMA, and exposed to either 24 hour dark, a light/dark cycle, or 24 hour light. After growth initiation, growth rate and degree of sporulation were measured. Results indicate that growth rate and degree of sporulation differ with light conditions and media. On CMA the initial growth rate under 24 hour light was higher than under light/dark and 24 hour dark. Light/dark conditions were more conducive to early sporulation whereas 24 hour dark delayed sporulation. Sporulation did not occur under any light condition on MEA for six days. The data indicate that under favorable growth conditions, S. chartarum can sporulate under any light condition. Therefore, S. chartarum growing in buildings in dark areas can sporulate for dissemination into the air.

Mycotoxin Spectra as a Biochemical Parameter for Occupational and Environmental Fungus Exposure

M. Müller, Ph.D., J. Bünger, M.D., E. Hallier, M.D., Prof. Center of Environmental and Occupational Medicine, Department of Occupational and Social Medicine, Georg-August-University e-mail: ehallie@gwdg.de

Mycotoxins are metabolites formed by molds in foodstuffs, fodder and organic waste materials. All molds produce specific mycotoxins and species can be characterized by their mycotoxin spectra. We have established a method for mycotoxin extraction from defined cultures. HPLC separation with diode array detection or iontrap mass spectro-metry and comparison to an authentic standards library was used to screen seven Aspergillus and Penicillium species collected at waste treatment plants for their major mycotoxins. The human hepatocarcinogen sterigmatocystin was detected in Aspergillus versicolor and Aspergillus nidulans. Verruculogen, a potent tremorgen, and fumagillin, formerly used as a cytostatic drug, are mycotoxins found in Aspergillus fumigatus. Penicillium crustosum and Penicillium brevicompactum each produced the tremorgens roquefortine C and penitrem A. The mycotoxin standards library can be used to identify specific mold species in ambient air samples from environmental and occupational investigations.

Membrane Toxic Substances in Water-damaged Construction Materials and Fungal Pure Cultures

University of Helsinki, Department of Applied Chemistry and Microbiology, Division of Microbiology,University of Helsinki, Finland Email: joanna.peltola@helsinki.fi

We showed toxic substances in and isolated toxin producing microbes from water-damaged building materials. The toxins were extracted from the building materials and microbial cultures in methanol and analysed using boar spermatozoa as test cells. The fungal genera isolated from the toxic materials were identified as representatives of Stachybotrys chartarum, Aspergillus, Alternaria, and Penicillium. We found that toxin from the building materials and from four fungal isolates paralysed sperm cell motility and damaged cell membrane at low concentrations (EC50 < 10 mg of methanol soluble solids ml-1 of extended boar semen). The toxic isolates were identified to Stachybotrys chartarum. The toxin from one Stachybotrys chartarum strain was partially purified. To our knowledge, this is the first demonstration of membrane damaging toxin and their producer fungi from the building material.

Different Methods to Characterize Moldy Buildings

Toivola Mika, B.Sc., Reiman Marjut, Ph.D.; Hyvärinen Anne, M.Sc.; Meklin Teija, M.Sc.; Nevalainen Aino, Ph.D. National Public Health Institute of Finland, Division of Environmental Health, Laboratory of Environmental Microbiology e-mail: mika.toivola@ktl.fi

Different methods were evaluated for their potential to show unusual microbial conditions in a building. The buildings studied were schools and offices. The buildings were inspected for visible signs of moisture by a civil engineer. Samples were taken from the air, surfaces and structures. Indoor air concentrations of viable microbes were higher in moldy than reference buildings. This difference could not be seen in the total counts of biological particles. In most cases, the concentrations of microbes on the surfaces were low. In some cases microbial concentrations were high in material samples although no contamination could be seen in surface swab samples from the same damage area. With one sample or one method alone, the conclusion of the mold problem in the building could not necessarily be drawn.

Comparative Studies of Collection Efficiency of Airborne Fungal using Andersen Single-Stage Sampler and Air-O-Cell Cassettes

Stella M. Tsai1, M.Sc., Chin S. Yang, Ph.D., Patrick Moffett, Andrew Puccetti, Ph.D., C.I.H. P&K Microbiology Services, Inc., Cherry Hill, NJ

The collection efficiency of airborne fungal matter using the Andersen single-stage sampler and Zefon Air-O-Cell cassettes was compared in this study. A total of 814 sets of samples were collected. The correlation coefficient (r) between these two methods was at 0.33 (p < 0.05) for total fungal matter and at 0.29 (p < 0.05) for Cladosporium. The correlation coefficient (r) between the total fungal and Cladosporium concentrations collected from Andersen air samples and Air-O-Cell cassettes was at 0.78 (p < 0.05) and 0.62 (p < 0.05), respectively. Stachybotrys-like spores were detected in 74 Air-O-Cell samples. Stachybotrys chartarum was detected in 5 Andersen samples. Four sets of samples showed Stachybotrys chartarum on both Air-O-Cell and Andersen air samples.

Trichothecene Mycotoxins in Some Water-Damaged Buildings

Tapani Tuomi1 Ph.D., Lauri Saarinen M.Sc., Sanna Lappalainen Lic. Phil., Outi Lindroos M.Sc., Marjo Nikulin Ph.D., Kari Reijula M.D., Ph.D. Finnish Institute of Occupational Health, Uusimaa Regional Institute, Helsinki, Finland, e-mail tapani.tuomi@occuphealth.fi

Bulk samples of moldy interior finishes, settled dust, contact inoculated microbiological samples (mixed cultures), as well as pure cultures isolated from indoor environments, were subjected to the qualitative and semi-quantitative simultaneous analysis of 12 trichothecenes. The analysis method was developed as a result of the present study and it includes extraction, sample pre-treatment and reverse-phase HPLC-separation with following tandem mass spectrometric identification and quantitation using electrospray ionization on a quadrupole ion trap mass analyzer.

Similarly to previous studies on fodder or foods, contaminated with trichothecene producing moulds, diacetoxyscirpenol and T-2 toxin were the most prevalent trichothecenes. Apart from these, 3-acetyl-deoxynivalenol, T-2 tetraol, verrucarol and roridin A were occasionally present, particularly in samples contaminated with Fusaria or Stachybotrys spp. Satratoxins G and H were also found on rare occasions, especially from sites with a severe occurrence of Stachybotrys spp. All examined sites were Finnish water-damaged buildings, with confirmed health implications, resulting from fungal propagation.

Immunochemical Detection of Mycotoxins Associated with Stachybotryotoxicosis

R. Dietrich, Ph.D., E. Johanning, M.D., M.Sc., M. Gareis, D.V.M., Ph.D., Prof., E. Schneider, Ph.D., E. Usleber, Ph.D., E. Märtlbauer, Ph.D., Prof. Institute for Hygiene and Technology of Food of Animal Origin, University of Munich, Munich, Germany e-mail: R.Dietrich@mh.vetmed.uni-muenchen.de

Using high-affinity monoclonal antibodies (Mab) against roridin A which exhibit cross-reactions with satratoxins and other macrocyclic trichothecenes several immunochemical methods were developed for the direct detection of satratoxins in contaminated building materials. Applying a lab-independent enzyme-linked immunofiltration assay (ELIFA) technique positive results were obtained within 10 min for toxin concentrations of > 200 mg/kg. The detection limit of a microtiter plate assay format was 1 mg/kg. For samples contaminated with Stachybotrys chartarum, an excellent agreement could be observed between the enzyme immunoassay (EIA) and a cytotoxicity test. The results of the EIA could be confirmed by HPLC analyses using immunoaffinity columns for sample clean-up. Furthermore, a new method was established for the sensitive detection of verrucarol in serum. In three out of 58 serum samples traces of verrucarol were detected. Two other sera reacted strongly positive for macrocyclic trichothecenes. Altogether, these results underline the importance and usefulness of immunochemical methods for epidemiological studies on airborne mycotoxins in indoor environments.

Mitigation of Visible Fungal Contamination in Buildings: Experience From 1993 - 1998

Philip r. Morey, P.H.d., Daryl Sawyer, B.s. AQS Services, Inc,. 2235 Baltimore Pike, Gettysburg, PA

Experience in the early 1990’s led to the recommendation that removal of visible fungal growth from interior surfaces in buildings be performed in a manner that minimized the dispersion of particles (dusts) in indoor air. Several documents beginning with the 1994 New York City Guidelines on Assessment and Remediation of Stachybotrys atra in Indoor Environments provided practitioners with procedures that can be used to remove visible fungal growth from building interiors. All fungal remediation guidelines recommend that sustained and extensive fungal growth on interior surfaces should be physically removed and that people performing remediation work should use appropriate personal protective equipment. In addition, all guidelines published in 1993-1998 recommend that moisture problems in building infrastructure be fixed in order to prevent new fungal growth. Fungal remediation guidelines specify a certain surface area of visible fungal growth (generally 3 to 10m2) that requires containment barriers similar to those used when hazardous chemical or physical materials are removed from buildings. Misunderstanding of 1993-1998 guidelines has resulted in both overly conservative approaches to clean-up as well as to dispersion of fungal spores throughout a building because of poor dust control. Unlike guidelines on removal of hazardous chemical and physical agents where rigid inspection protocols and specific numerical guidelines are appropriate, the removal of mycobiota is a variable process depending on many factors including the biology of the fungal taxa. Factors such as the following should be considered during the remediation process: (a) the location, extent, and kind of fungal growth in building systems, (b) the susceptibility of building materials to biodeterioration, (c) the porosity of building materials, (d) the susceptibility of occupants to bioaerosol exposure, and (e) sampling and monitoring protocols appropriate for the fungal contaminants. Fungal remediation in buildings continues to require a considerable degree of professional judgement with regard to procedures appropriate for containment of dusts and for control of the contaminant mycobiota.

Microbes and Moisture Content of Materials from Damaged Building

Meklin T., M.Sc., Haatainen S., B.Sc, Kauriinvaha E. M.Sc, Kettunen A-V. M.Sc, Haverinen U., M.Sc., Vahteristo M. M.Sc., Viljanen M. Prof., Nevalainen A Ph.D. National Public Health Institute, Kuopio, Finland, e-mail Teija.Meklin@ktl.fi

The aim of the study was to characterize the microbial flora of the envelope of a school building and the correlation of the microbial concentrations and the moisture content of materials was estimated. Material samples (n=95) were taken from different structures before the dismantling of the school. In all, 40 sample pairs were taken from which both concentrations of microbes and moisture contents (% by weight) were determined. The range of the total concentration of fungi was <45 - 8 600 000 cfu/g for all the analyzed material samples. The growth of microbes in a building is mostly regulated by the moisture of the materials and often the elevated concentrations of mesophilic fungi (>10 000 cfu/g) were associated with elevated moisture content of the material. However, the microbial concentrations did not fully correlate with the moisture contents of the material.

Concentrations of Viable Spores of Fungi and Actinomycetes in Ventilation Channels

Outi Lindroos, M.Sc., Sanna Lappalainen, Phil.Lic., Kari Reijula, M.D, Ph.D. Uusimaa Regional Institute of Occupational Health, Indoor Air & Environment Program, Finnish Institute of Occupational Health, Helsinki, Finland, E-mail: Outi.Lindroos@occuphealth.fi

In this work we studied if accumulation of fungal spores to the ventilation channels has any significance as microbial source in indoor air. Concentrations of viable spores were determined from samples that were collected from ventilation channels, and the geometric mean and median values were calculated. The concentrations of fungal spores and actinomycetes on ventilation channel surfaces were low even in abundantly dusty channels, and therefore accumulation of spores in the channels did not seem to form any significant microbial source in indoor air. In the samples from exhaust channels in buildings with verified mold damages, prevalence of fungal species such as Acremonium sp., Aspergillus versicolor, Chaetomium sp., Eurotium herbariorum, Paecilomyces variotii, Phoma sp., Trichoderma viride and Ulocladium sp. was better indicator for damages than the concentrations of fungal spores.

Air Quality Restoration in a Fungal Contaminated Building

Herman Sabath M.P.H., Ph.D. International Environmental Diagnostics, Inc., New York 

Attempts to mitigate microbial contamination of a four story office building by use of common standard cleaning procedures proved inefficient by post abatement analytical results. Toxigenic, allergenic and pathogenic fungi were identified in sampling evaluation as a follow up to microbial abatement. Microorganisms such as Stachybotrys, Aspergillus, Penicillium, Cladosporium and Fusarium were detected in air and surface samples of a four story office building. Review of the chain of events revealed that microbial recolonization, cross-contamination and recontamination had occurred at this facility.

IED, Inc. an environmental company specializing in microbial abatement and air quality restoration in indoor environments was commissioned to the project of biocidal clean-up and air quality restoration. Success of such projects are strictly dependent on applied scientific knowledge and experience. IED, Inc. successfully completed the biocidal and air quality restoration project as proven by post abatement analytical results performed by an independent third party monitoring company and laboratories.

Sampling, Results & Remediation in 300 "Sick Houses"

Jeffrey C. May, email: jmhi@cybercomet

Inhabitants in "sick house syndrome" (SHS) homes suffer from allergy, asthma, sinus and other respiratory problems, including hypersensitivity pneumonitis and aspergillosis. Reports from 300 SHS homes were compared to randomly-selected reports from 150 homes inspected as part of pre-purchase agreements in the Boston area. SHS homes were almost twice as likely as other homes to have forced hot air heat, central air conditioning and finished/carpeted basements. Elevatedlevels of mold were found in 74% of SHS homes. The largest sources of bioaerosol were found to be carpeting; heating, ventilation, and air conditioning (HVAC) equipment; and beds and sofas. Respirable "carpet dander" from damaged wool carpet fibers can be an irritant. Effective SHS remediation may include: carpet removal; thorough coil /duct/blower cleaning along with replacement of contaminated fiberglass lining materials in HVAC equipment, and cleaning fleecy items with dry (super-heated) steam. Mite-barrier mattress and pillow covers are always recommended. SHS may be a cause for increased asthma rates.

Identifying and Preventing Fungal Contamination Problems in New Home Construction

Päivi Salo, M.Sc. University of North Carolina at Chapel Hill, School of Public Health, Department of Environmental Sciences & Engineering, E-mail:psalo@mindspring.com

The objectives of this study were to identify construction materials and practices prone to introduce fungal contamination into new single family homes, and to identify preventive maintenance procedures which limit fungal colonization and amplification. This study was performed over a two year period in North Carolina. Eight construction sites were observed during construction and during the first year of occupancy.

Building materials were improperly stored and exposed to rain and/or high humidity. Inadequate drainage caused many crawl spaces to remain damp. Many HVAC systems were inadequately sealed, enabling rainwater and construction debris to enter the systems, and thereby providing suitable fungal substrates. Negative pressure within the building envelopes caused air infiltration from the crawlspaces. Many of the observed problems found could be avoided by educating builders and homeowners. Early identification and prevention of potential fungal reservoirs and amplification sites is preferable to costly analysis and repairs afterwards.

A Toxic Mold Cleanup Guide

Jim H. White, B.A.Sc., P.E.O. CMHC National Office, Ottawa, Canada

Many houses and small buildings are moldy, and some have extensive growth of toxigenic molds that can cause health problems at lower exposures than the phyloplane molds that are more common outdoors. Canada Mortgage and Housing Corporation, the Canadian federal housing agency, has performed a great deal of research into moldy houses and has created a number of publications on mold avoidance and cleanup. This document is one of a new series that addresses toxic mold cleanup as well as solving moisture problems and avoiding mold growth in the first place.

Molds as an Environmental Factor in Infant Leukemia?

Casteleyn L, Van Damme K, Van den Berghe H. Center for Human Gentics, University of Leuven, Leuven, Belgium

The possible influence of occupational, environmental and lifestyle exposures of parents on the occurrence of infant leukemia is studied. Infant leukemia diagnosed within the first 6 months of life occurs at a frequency of around 1 case in 50.000 live births and shows a consistent genomic defect involving the 11q23 band (the MLL gene). These rearrangements are considered to offer circumstantial evidence for an environmental factor being involved and compelling evidence exists that the particular mutation is acquired in utero.

Mechanisms of Adverse Health Effects of Moldy House Microbes: in vitro and in vivo studies on toxic effects and inflammatory responses

Maija-Riitta Hirvonen, Ph.D., docent, National Public Health Institute, Division of Environmental Health, Kuopio, Finland, e-mail: maija-riitta.hirvonen@ktl.fi

Epidemiological evidence shows that building moisture and microbial growth are associated with respiratory symptoms related to inflammatory reactions, ie. irritation, infections and asthma. At present, it is not known which are the most important causative microbes able to induce these adverse effects, what are the specific cellular effects and, particularly, what are the mechanisms of them. These data are, however, needed for proper risk assessment of the moldy house problem and the measures taken to solve it. There is an urgent need on experimental work on cell cultures and laboratory animals with the microbes isolated from moldy buildings suspected to be harmful. Such data is at present to most extent missing but the present plan is aimed to provide it comprehensively.

We have recently observed that 1) streptomycetes induce production of inflammatory mediators i.e. nitric oxide (NO), cytokines and reactive oxygen species (ROS) and cause cell death in mice macrophages in vitro, 2) these responses are not dependent on the viability of the spores of streptomycetes, and preliminary: 1) growth conditions play an important role in the ability of these microbes to induce the production of inflammatory mediators and to cause cytotoxicity 2) streptomycetes produce NO and cause cytotoxicity also in human lung epithelial cell line, and 3) the strains of the streptomycetes active in vitro also elevate the same inflammatory mediators in bronchoalveolar lavage fluid (BAL) in rats after an intratracheal instillation to lungs. Altogether, these results suggest that certain moldy house microbes are able to induce inflammatory responses and/or to cause cell death in mammalian cells. This may play a central role in the cascade of events leading to the adverse health effects.At this phase, it is inevitable to study which other microbes characteristic to moldy houses cause similar effects and what are the effects of these microbes in lungs.

The overall aim of the study is to find out which microbes among the mixed population of the microbes present in the moldy houses are able to cause adverse respiratory health effects and what are the mechanisms of them. The focus is on inflammatory responses and cytotoxicity in human and mice cells and local toxicity in lungs and effects on respiration in animals. Effects of six typical microbes isolated from moldy buildings are studied: Streptomyces anulatus, Sreptomyces californicus, Aspergillus versicolor, Stachybotrys atra, Fusarium, and mycobacteria. This plan evaluates effects of these microbes in vitro in both human and mice cell cultures, deepens our previous work with streptomycetes to new mechanisms and expands studies to animals in vivo. The specific aims for the in vitro studies are 1) to study in detail the effects and the mechanisms of cell death and inflammatory responses in human and mice macrophages, induced by these microbes and their combinations, 2) to study cytotoxicity and the inflammatory responses induced by these microbes in human lung epithelial cells 3) to study the relation between growing conditions of the microbes and their ability to induce inflammatory responses and cytotoxicity. The specific aims for in vivo studies are 1) to study effects of streptomycetes and the microbes proving to be harmful in in vitro studies in lungs of mice after intranasal instillation (inflammation, local toxicity in lungs), 2) to identify the target cells of effects in the airways 3) to evaluate the effects of the microbes on respiration in guinea pigs after intratracheal instillation. This study identifies potentially harmful microbes present in moldy houses to cause respiratory effects, describes those effects in the lungs of laboratory animals and elucidates the cellular mechanisms of moldy house effects. These data will form a new fundamental basis for risk assessment of the health effects of those microbes and help to develop methods for biomonitoring of harmful microbial exposure. Identification of the most harmful microbes is also the basis for decisions to solve the moldy house problems. In vitro studies: to investigate the effects induced by occupational exposure to microbes present in moldy houses on nasal functions and production of inflammatory mediators in nasal lavage fluid (NAL) cells in healthy and symptomatic subjects.

Exposure to Bioaerosols

Principal investigator: Aino Nevalainen, docent, Ph.D. National Public Helath Institute, Laboratory of Environmental Microbiology, e-mail aino.nevalainen@ktl.fi

Moisture and mould problems of buildings are associated with respiratory symptoms and diseases. The association between the building damage and the adverse health effects is well known, but little is known about the mechanisms of the diseases and about the actual exposure causing these health effects. The exposure has been characterized in many indirect methods. It has been shown that concentrations of viable fungi and bacteria and microflora of the indoor air in a damaged building differs from that of a normal building. However, viable microbes only comprise about 10% of the total number of biological particles in indoor air, and thus are a proxy of the real microbial exposure, the nature and quality of which is largely unknown so far. Toxic components derive from fungi and bacteria growing in building materials, but it has not been shown how the exposure to these components via indoor air takes place. The principal aim of this study is to find out whether the exposure to bioaerosols and fine particles of individuals with symptoms typical to mould exposure differs from that of matched control individuals. The detailed aims are:

1. To study whether the exposure to viable microorganisms, total number of biological particles and fine particles of the individuals with mouldy house symptoms differ from the exposures of matched control individuals with no such symptoms

2. To study whether the same inflammatory mediators which are detected in the nasal lavage fluid of exposed individuals, can also be detected in vitro in the cell culture medium of macrophages after the exposure to particles, collected during the exposure period.

3. To study the within person and between persons variation of exposure to bioaerosols using repeated measurements.

4. To compare results of personal exposure and stationary sampling measurements in home and work.

A group of 35 individuals with symptoms typical to mould exposure will be selected and a control individual with no such symptoms will be selected for each index person. A 24-hour sample collection for bioaerosol and fine particles will be made with personal and stationary sampling in homes and in the working places. The homes and working places will be surveyed for signs of moisture damage according to a check list. During the sampling period, diaries on time activity in different microenvironments and on symptoms will be filled and PEF will be recorded. A nasal lavage fluid sample will be taken from each individual after the sampling period. This sampling period will be repeated twice with each individual.

Bioaerosol and particle sampling is made with button samplers developed in the University of Cincinnati.The concentration of collected particles will be analyzed gravimetrically. Viable microorganisms of filter are cultured and the total number of biological particles counted with an epifluorescence microscope.

Toxicological studies: A nasal lavage fluid (NAL) sample will be collected from each individual after the sampling period. Production of inflammatory mediators (NO, cytokines: TNFalfa, IL-1, IL-5, IL-6, IL-10) in the NAL cells will be analyzed.

Schools, Mould and Health - An Intervention Study

Principal investigator: Aino Nevalainen, docent, Ph.D. National Public Helath Institute, Laboratory of Environmental Microbiology, e-mail aino.nevalainen@ktl.fi

Moisture and mould problems in school buildings cause exposure to biological indoor air pollutants for the school children, teachers and other personnel, resulting in respiratory symptoms and infections and in some cases, even increased incidence of asthma. A number of occupational diseases caused by biological indoor air pollutants have been recently recognized among teachers and personnel of moisture -damaged schools. Indoor air quality and health problems are common, and the municipalities are putting remarkable resources on the repair measures but little documented data is so far available about the effects of the repair measures on the users health and thus on the cost effectiveness of the repairs. This intervention study is designed to increase our understanding about the effects of the repair measures on the school students health and exposure. The overall aim of the study is to find out whether the moisture and mould repairs of the school buildings have an effect on the exposure to indoor air pollutants, and on the prevalence of respiratory symptoms and diseases of the school students Both the health status and exposure are measured in detail before the repairs are started, and the measurements will be repeated after the completion of the repairs. The study cohort consists of elementary school children and high school students (N=2500). The detailed objectives are:

1. To find out if there is a dose-response effect depending on whether an individual is exposed to mold in school or home or in both,

2. To find out whether the elimination of microbial growth and repair of moisture damage will eliminate the increase of respiratory symptoms and infections among the school students,

3. To find the most relevant methods to characterize the exposure associated with building moisture and mould, and

4. To develop a method to monitor the toxicity and biological activity of the indoor air particulate material.

Microbial exposure is characterized with microbial samples from the indoor air (impactor and filter), surface swab samples, house dust and pieces of damaged materials. Ergosterol concentration and lipid fatty acid profile by GC; acute cell toxicity and other biological activity, see the project of Dr. Hirvonen; concentrations of dog and cat allergens and house dust mites from house dust. The buildings will be inspected for their signs of moisture according to a chekc list and the repair measures will be documented. Health status of the children will be monitored with questionnaires which will be validated with absence analysis and symptom diaries.

This study is linked with the follow-up of the health of the personnel, the technical monitoring of the effectiveness of the repairs, (financed by the Finnish Fund of Occupational Health and Tekes), and several projects of the Consortium studying the exposure to bioaerosols and mechanisms of the health effects. The results will provide tools

for assessing the effectiveness of the repairs in decreasing the symptoms

for understanding which types of damage/exposure produce the highest risk to health

for monitoring the indoor exposure to biological contaminants

for monitoring the indoor air exposure to biological contaminants

for assessing the urgency ranking of the repairs, needed in the community planning

Development of Methods to Monitor the Success of Repair Measures

National Public Health Institute, Division of Environmental Health,

Laboratory of Environmental Microbiology, e-mail: aino.nevalainen@ktl.fi

Moisture and mould problems have been assessed as a major problem in the modern building stock, and their repair and prevention are one of the most important challenges in the research of building technology. As these problems often lead to health complaints among the users, the prerequisite of a successful repair process is the identification of the ultimate causes of the moisture accumulation and the development of reasonable repair methods. This work must go in parallel with the health-related research including the prevention of diseases and wellbeing of the occupants. Suitable methods for the follow-up of the success of the repairs are urgently needed. The technical criteria of the repair planning may somewhat differ from the health-based criteria. For example, a material previously contaminated with mycotoxins may still be technically usable, but not acceptable for health-based reasons.

The aim of the study is to develop method for monitoring the success of moisture and mould repairs. The strategy is to combine the technical and health-based approach. The material for the study will be collected from large repair projects with an intervention-type studyesign. The exposure and health status of the occupants will be followed throughout the process and the technical repair solutions will be carefully documented.

Neurotoxic Effects of Microbial Toxins

Principal investigator: Prof. Kai Savolainen, MD, PhD, Finnish Institute of Occupational Health, Industrial Hygiene and Toxicology, e-mail: kai.savolainen@occuphealth.fi

In Finland, half of all homes have a moisture problem, and microbial growth occurs in 20 percent of the cases. Altogether 500 000 individuals are annually exposed to indoor bioaerosols. Recent evidence suggests that this exposure may cause CNS effects in addition to adverse effects in the respiratory system. In fact, adverse CNS effects after exposure to bioaerosols, i.e. bacteria, fungi, molds, microbial cell wall components or metabolic products of microbes, have been found in experimental animals and humans. Available evidence indicates that both neuronal and glial cells can be affected by this exposure. The effects of microbial toxins on the CNS are of particular importance because even a small excess morbidity of brain diseases may imposes a major burden on the health care system and the society at large. The focus of this research is to increase understanding of the role of microbial toxins in bioaerosol-induced CNS health effects. The overall objective is to study the mechanisms whereby LPS, and toxins of Fusarium multiforme, fumonisin B1, and of Stachybotrus chartarum (trichothecenes) activate neuronal and glial cells. The specific aims: 1. To study cell activation by the toxins; 2. To study altered gene expression by these toxins; 3. to study mechanisms of apoptosis induced by these toxins; and 4. to explore the role of cytokine production in the effects of these toxins. Methods: ROS and NO production will be studied with a fluorescent probe, and photometrically. Cell death will be analysed fluorometrically, and apoptosis with DNA fragmentation and with a fluorescent probe. Transcription factor binding will be studied with an EMSA assay, and cytokine production with an ELISA assay. Expression of iNOS is explored at protein, and of an apoptosis promoter BAX, and an antiapoptotic gene Bcl-2, at mRNA level. Also caspase expression (mRNA level) and activity will be studied. We expect that the proposed studies will provide valuable information of the mechanisms of effects of microbial toxins in neuronal and glial cells. This information can be utilized in assessing health hazard of bioaerosol exposure, and in evaluating the need of human epidemiological studies.

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The Biological Activities of the Metabolites of Microbes Present in the Indoor Air

Principal investigator: Prof. Atte von Wright, Ph.D., Department of Biochemistry and Biotechnology, University of Kuopio, e-mail: atte.vonwright@uku.fi

Abstract Microbial aerosols in houses suffering from excessive humidity are a serious public health problem. So far the main attention has been focused to the sensitizing properties of microbes and their degradation products. However, the microbes present in problem houses (molds and actinomycetes) are known producers of bioactive secondary metabolites such as antibiotics and toxins. The role of these secondary metabolites in the sick building syndrome is still largely unknown. In a previous screen a number of fungal and actinomycete isolates from problem houses were shown to have antibiotic properties against other microbes. Some of the isolates produced unidentified compounds reacting with DNA. Because of the role of DNA-damage in tumour induction these genotoxins might present a previously unknown risk associated with indoor air. In order to evaluate this risk it is necessary to identify the compounds in question, study their genotoxic potential, their production conditions and prevalence, volatility, stability, and possible other harmful effects. The aim of the project proposed in this application is to isolate and identify some of the most potent genotoxins produced by the representative microbial isolates from problem houses. The isolation is based on the fractionation of the culture media by standard procedures (organic solvent extraction, affinity chromatography, thin layer chromatography, HPLC etc.). The activity of the fractions is monitored by their selective toxicity to a DNA-repair-deficient bacterial strain extremely sensitive to DNA-damage. A more thorough mutagenicity testing with both bacterial and mammmalian test systems is then applied to the purified fractions. Purified substances are characterized using mass spectrometry, other spectroscopic methods and NMR. When sufficient information of their chemical nature has been obtained  this can be used as a basis of their detection in actual problem houses and for a tentative risk evaluation.

Environment, Decision-Making and Well-Being - Insecurity, Uncertainty and Crisis of Expertise

Principal investigator: Prof. Aulikki Nissinen, University of Kuopio, Department of Public Health and General Practice, email: aulikki.nissinen@uku.fi

The project examines the association between perceived insecurity and uncertainty of people and environmental problems (mould and asbestos) theoretically affecting health, creates with local population research based model for decision-making process to control the problem. The project first makes baseline measurements on indoor air quality, asbestos in yards, health profile of population and perceived insecurity and uncertainty among the population as well as the awareness and knowledge about the problem. Based on these results and analysis of current decision making system the model for decision making is created by researchers, decision makers and laypopulation. The model is also tested.

Specific aims are:

to determine microbiological quality of indoor air in public buildings including schools and the health status of the users of buildings, to measure asbestos of soil in the intimate surroundings of people living in the asbestos polluted area (Tuusniemi), to compare health profile of population with the Finnish population, to assess insecurity due to and awareness of the environmental threats in the area, and the knowledge about the problems among the population, decision makers and municipality officers, to assess uncertainty about expertise among population and degree of uncertainty among decision-makers and civil servants about decision-making process, to create and test with researchers, decision-makers and laypeople the model for decision making process based on the analysis of the current decision making and the results of the measurements, to measure change in insecurity, uncertainty, awareness and knowledge among the population, decision makers and the officers of the municipality after evidence based information campaign.

Radonsafe Foundation, Moisture Prevention and Air Exchange in a Healthy Building

Principal investigator: Hannu Arvela, Head of laboratory, Radiation and Nuclear Safety Authority, e-mail: hannu.arvela@stuk.fi

The study aims at combining the construction of a good radon-tight foundation and moisture prevention in new low rise residential houses. The study aims also at developing the applicability of different ventilation strategies, the control of depressure in dwellings and use of fresh air vents for reduction of indoor radon concentration. The study also deals with the radonsafe foundation construction of blocks of flats. Without any radon prevention the recommended limit for new houses, 200 Bq/m3 will be exceeded in 50 % of new houses in wide areas of the southern Finland. The recommended radonsafe construction of a slab-on-grade foundation is based on the use aluminized bitumen felt which seals the foundation and prevents the leakage of radon-bearing soil air into living spaces. The construction provides also a qualified moisture prevention against moisture originating from the subsoil. Radon safe construction will be compared with the normal moisture prevention, using simulation calculations. In some houses the moisture of house constructions and subsoil will be measured.

The study will be carried out in 15 single family houses where the radon safe constructions will be controlled by experts of this project. Indoor radon concentration, air exchange rate, depressure and the operation of air exchange instrumentation will be measured carefully. This provides a basis for estimating the radon entry rate into the houses and the success in radon prevention. The direct influx of radon-bearing soil air increases also the indoor radon concentration of flats of the lowest floor dwellings, in some cases also in upper floors. In this study the alternative radon foundation constructions of apartment houses will be surveyd, measurements will be made in 5 test houses. The depressure in flats has also a remarkable effect on indoor radon concentrations. The applicability of fresh air vents in flats for reduction of depressure, radon concentration and generally the indoor air quality will be studied in test houses and laboratory. The effect of commercially available fresh air vents on depressure and draft will be studied.

Adsorption, Desorption, and Chemical Reactions in the Particulate Matter Collected on Air Filters and Ducts

Principal investigator: Prof. Pentti Kalliokoski, Department of Environmental Sciences, University of Kuopio, e-mail: pentti.kalliokoski@uku.fi

Even though air handling systems are intended to improve indoor air quality and climate, they have often become major sources of odorous compounds. The filter has usually been the main cause of sensory pollution, and its emission has increased with time. Dirty ducts and coils have also remarkable odor emissions. Already the new ducts are often heavily contaminated with processing oil residues and with dirt accumulated during storage and construction period of the building. On the other hand, it has been found that the sum of the odors released from various components may not be in good agreement with the perceived emission from the whole system. Adsoption and desorption processes between accumulated particulate impurities and gaseous pollutants seem to play an important role for the final air quality. In addition, chemical reactions between oxidizing pollutants, such as ozone, and the organic pollutants adsobed on particles have been suggested to occur and to further deteriorate the air quality.

In this study, the adsorption and desorption properties of dust collected on filters and other parts of the air handling units are investigated. Especially, the significance of adsorption and desorption phenomena on air quality is considered. The data are also used to model the interactions between particulate matter and gaseous compounds. The chemical reactions will be studied. The ultimate aim is to prepare guidelines for cleaning and maintenance.

Dust samples will be collected from office buildings locating in downtown areas of Helsinki and Kuopio. For comparison, samples will also be collected from buildings in clean suburban area in Kuopio. The properties, such as spesific surface area, density, carbon content, and carbon/nitrogen-ratio, affecting the adsorption/desorption properties will be determined. Used air filters will be installed into a laboratory scale air handling unit for further studies. The gaseous compounds that will be investigated include the common volatile organic compounds belonging to aliphatic hydrocarbons, aldehydes, terpenes, and aromatic compounds. In addition to the physico-chemical properties, the odor emissions from the dust samples and the effects of ozone will be determined.

Fungal Allergens and Antigens - Their Characterization and Biological Effects in Mice after Inhalation Exposure

Principal Investigator: Anna-Liisa Pasanen, PhD, docent, University of Kuopio, Department of Environmental Sciences, -mail: annal.pasanen@uku.fi

The non-allergic symptoms that are reported by people occupying moldy buildings are assumed to be caused by microbial metabolites and components, such as fungal b(1‹3)-D-glucans, even though the biological effects of these agents and their relationship with fungal antigens have not yet been clarified sufficiently. It is also possible that the reactions that are reported to be caused by fungal antigens might be mediated by mechanisms other than those of immediate type of allergy. In this context, it is interesting that the molecular structure of only few fungal allergens is known so far, and that the cross-reactivities between different fungal species have been weakly characterized. On the other hand, skin and serological tests are generally performed in the clinical work with unstandardized fungal extracts without the clear comprehension, how the results should be interpreted. The specific knowledge on the composition of fungal allergens would also be useful for the development of specific detection methods so that the most harmful fungi, e.g. Stachybotrys chartarum, could be easily identified in the environment.

This research project is divided into two parts. In Part 1, Characterization of allergenic components of some mold species and the development of a specific detection method for Stachybotrys chartarum, the antigenic compositions of four to six fungal species (S. chartarum, A. versicolor, P. brevi-compactum, C. cladosporioides, and two yeasts) the exposure to which is common in moldy buildings or agriculture, is characterized by SDS-PAGE and immunoblotting using immune and human sera. The cross-reactivities of the fungi between each other and with other common fungal species are investigated by the same method, and the specific components are determined. Polyclonal and/or monoclonal antibodies are created against these components. ELISA methods for measuring mold-specific antibodies and ELISA inhibition methods for measuring the antigenic components of these fungi are also developed. The important objective of the study is to produce the specific components of S. chartarum and possibly of some other fungus as recombinant proteins. For identifying these proteins, the cDNA library is created and the library is screened by specific antibodies. The gene coding for the specific component is transformed in the Pichia pastoris yeast for production. This approach allows the definition of the nucleotide and amino acid sequences as well as the production of the protein in great amounts and, thus, enables the development of a rapid detection method for the fungi, particularly for S. chartarum.

In Part 2, Exposure of mice by inhalation to fungi, irritating effects in the respiratory tract and immune responses, mice sensitized to the fungi mentioned above, are repeatedly exposed to various amounts of fungal antigens, glucans and volatile metabolites by inhalation, and the respiratory functions of mice are monitored continuously during the experiment. The irritating potencies of the agents are determined. After the exposure, the levels of specific antibodies (IgG, IgE, IgA) in serum and inflammatory mediators (IL-1, IL-6, TNF-a) in bronchoalveolar lavage fluid are measured. Histological analyses are performed from the tissue samples of the upper and lower airways. According to the results, the biological effects of fungal agents can be elucidated.

Indoor Air Quality Control

Principal investigator: Kristina Saarela, M.Sc.,VTT Chemical Technology, Environmental Technology, E-mail: Kristina.Saarela@vtt.fi

Population exposure and to air pollution caused by different chemical compounds and to annoying odours inside buildings is usually much more significant than outdoors due to higher concentrations and overall longer periods of time spent indoors. As the indoor air quality (IAQ) has a non-negligible impact on human health and comfort, a healthy indoor climate should be one of the main objectives of today's construction practise. This, however, requires development of comprehensive and effective ways for characterisation of the air indoor and related symptoms. Until now, only a few health effect studiesconcerning the health and discomfort effects of indoor air have been carried out. Indoor air is strongly affected by the surrounding building materials releasing a wide variety of chemical compounds such as volatile organic compounds (VOCs), aldehydes and ammonia, which are suggested to cause several types of health effects and discomfort. As only little knowledge of material emissions and their health and acute discomfort effects is currently available and as there is no consensus on the harmful chemical compounds, source emission control is currently considered the most effective control option. Some classification systems, such as Finnish 'Classification of Indoor Climate, Construction, and Finishing Materials' are already been promoted. The development and use of new healthy materials however requires deeper understanding of material emissions and their correlation with perceived indoor air quality, personal health and well being.

The first aim of this research is to establish the causal connection between the diagnosed health effects and the chemical composition of indoor air. Based on this knowledge, criteria for good indoor air may be created. The second significant aim is to generate more health and comfort related IAQ and material emission evaluation procedures to be utilised in indoor air diagnostics and material classification purposes. These aims can be achieved by the following research tasks:

simultaneous production of health effect and IAQ data

understanding of causal connection of both indoor and outdoor emissions and their impacts on personal health and well-being through modelling and with the help of an extensive database

development of comprehensive chemical measurement and sensory evaluation techniques, complementary to currently used ones, for IAQ and material emission control

implementation of new testing procedures for indoor air and material emissions

prediction of IAQ from material emission data with the help of a model, which takes into account also the physical

parameters, e.g. ventilation rate and sorption effects

The first task is aimed at establishing criteria for healthy indoor air. This is obtained by combining the existing data from

VTT's IAQ-database, the indoor air data from EU-Expolis study and comprehensive health related data, which is obtained by choosing subjects among the patients treated in Helsinki University Central Hospital because of building related symptoms. The health and comprehensive indoor air data obtained from the subjects and their homes are compared with similar data of a control group. For collecting the comprehensive indoor air data new methods are utilised in order to detect very volatile (VVOCs) and semi-volatile organic compounds (SVOCs) and polar compounds.

The aim of the second task is to develop procedures to evaluate the irritating and odorous chemical compounds of material emissions and the perceived air quality. The causative relationships between sensory assessment method used in the present Finnish Classification of Finishing Materials, olfactometry and emission measurements in chemical terms are determined. The irritate data is produced in a study "Irritating properties of emissions from building materials" co-ordinated by University of Kuopio. The data obtained in this study is combined with data from VTT's DAME database in order to create an evaluation method for the revision of the present Finnish Classification for Finishing Materials.

The third task is focused on establishing a modelling procedure for predicting IAQ based on material emission data. In order to create an IAQ model, a procedure for testing sorption effects in laboratory scale is established and material emission together with sorption data is produced.

Mold and Moisture Transfer in Building Structures and Buildings with Particular Regard to the Prevention of Health Hazards

Principal investigator: Prof. Olli Seppänen, Helsinki University of Technology, HVAC-Laboratory, E-mail: olli.seppanen@hut.fi

The aim of the research project is to work out calculation facilities for mould transfer through constructions and inside the buildings, and to produce solutions and basis for solving mould and moisture problems by using pressurisation and insulation techniques with particular regard to moisture and mould transfer and health aspects. The following tasks are included in the research. 1. To work out mould transfer models by which both the spreading of the metabolic products and the mould spores in the constructions and inside the building can be modelled. 2. To give guidelines and limits on how to use pressurisation and insulation as repairing measures for preventing mould transfer. 3. To analyse possible side effects, moisture behaviour of constructions and mould growth caused by changing pressure conditions and, respectively, to produce guidelines with regard to moisture behaviour and mould growth. 4. To develop a new serum IgG avidity test in order to estimate the exposure to moulds in cross-sectional and follow-up studies.

Theoretical models for estimating the risk of occurrence of mould growth, growing and spreading of the mould and models for the moisture transfer will worked out. Models for spreading of mould will consider both particles (mould spores) and gases (metabolic products). Moisture transfer models will include the modeling of moisture convection and diffusion, but not water contact. Conditions for mould growth and material properties effecting on mould growth that are initial parameters for the estimating the mould growth, are mostly taken from other studies and literature. Laboratory tests and field measurements (in single-family and apartment houses) are used for developing and validation of the models. All developed mould and moisture models can be simultaneously used with traditional heat transfer and air flow models in modular simulation environment IDA.

Health hazards caused by microbes and water damages are studied by measuring the exposure of residents in test houses with new clinical methods. In addition to measuring the exposure, the purpose of clinical methods used is to estimate the time period, when the exposure to mould allergens has occurred. The state of residents' health is studied with epidemiological examinations. The same medical examinations are made before and after repairing measures and also in "clean" houses.

The behaviour of repairing solutions, worked out in the research, will be shown with computer simulations by using developed models and with laboratory and field measurements. The effect of the overpressurising will be analysed by studying the drop of contaminant concentrations, caused by changed direction of air flows (leakage), and also the change in moisture behaviour, caused by possible moisture convection, will be studied. In addition to measuring moisture conditions, the behaviour of mould growth will be studied by laboratory measurements and computer calculations. The important properties of constructions that make it possible to use the pressurising will be given and construction solutions where pressurising cannot be used will be outlined by examples.

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